Longer route with steady speed vs. short stop n go route
 

I've got 2 routes I can take to work, the first is 11 miles and starts out with a nice gentle 2 mile slope from about 500' to around 50', after that the route is full of undulations in the grade of the road, and a fair deal of stop lights. The last mile or so goes from 50' up to 400' and is quite steep but unavoidable no matter what route I take. The speed limit is 35 the whole way and it's pretty heavily trafficked on my drive in at 1:30 in the afternoon. There's been ongoing utility work and repaving for some time at night so I've avoided it for the most part.
Route 2 is I-5 and the I-205 belt route. Its considerably longer at 17 miles but traffic is always moving and the speed limit is 55 for the first half so I don't irritate many people. The I-205 stretch goes up to 65 but trucks are limited to 55 so I generally hang in the right lane and let them act as wind breakers ;)
I've done some very basic measuring of each route on a tank to tank basis but it's not really the most accurate since I do drive more than just to work and back. At least from what I can tell the freeway yields much better mpg but it's also a longer stretch. The road on the other hand is pretty unpredictable and also includes 2 railroad crossings.

You need a OBDII gauge! It makes driving fun and you will know for sure.

I would take the longer route and keep to the right lane. And around 40-45MPH as much as possible in your automatic. Top speed 60MPH. (don't fight the wind)

I wouldn't take the red-light route unless you can time the lights and hit all green.
(almost impossible, around here at least)

When I get in my car and drive somewhere I try my best to keep it moving until I get to wherever i'm going.

I've got some tax return money earmarked for an ultragauge :)
The lights are on timers through one town, then the rest are all sensors. I get lucky for the most part at night since there's hardly any traffic when I'm off work at 10:30 but the construction has been a real slow down lately.

I'd still consider the shorter route. Even if you get much worse fuel economy, its just that much shorter. You said you just got 26 mpg on your last tank. If that was taking the shorter route, you'd need to get over 40 mpg on the long route to make up for the additional gas usage. Now, this is possible, but as dirtydave said, you need a gauge to tell you for sure.

Stoplights and 35 mph limits are a hypermiler's playground. Rock that short route!

Don't listen to me, those guys are right! I changed my mind!

In addition to the fuel consumption, you also need to consider the wear and tear on the car's engine, transmission, brakes, and tires from driving either route. The shorter route with its stop and go driving and having to run up through the gears multiple times during each trip would produce more wear on the car's equipment. The longer route with its fairly constant speed and mostly low engine loads would be better for car longevity.

If you get 32 MPG on the freeway route and 26 in the city route, you should be using the same amount of gas, since the freeway is 6 miles longer, you need 6 more MPG to make up for the gas. Another thing to account is how the construction and railroads will affect your gas mileage. In my city, whenever there is a train, you will be sitting for at least 5 minutes, which is a pain in the butt and can cause excessive idling. Even if the city route for me is shorter, that doesn't make if faster or as fuel efficient.

Something that I have noticed about myself when driving around town is that I get aggressive and impatient in city driving, but on the freeway I get calmer and more patient. Find out which of these areas you are better at and stick to doing that. Most hypermilers try to stick to city streets because they can drive without the engine in those environments, but when driving an automatic you don't get as many opportunities to turn the engine off.

For what basjoos said, I think makes a lot of sense. Taking into account the load on the engine and transmission, stoplight to stoplight driving is pretty bad.

Like others have said, get instrumentation and track your statistics.

Sorry, not trying to be a jerk, but this math isn't correct.

11 miles @ 26 mpg = .42 gallons used

17 miles @ 32 mpg = .53 gallons used

These are not the same amount of gas.

To get the equivalent mileage you take:

.42 gallons / 17 miles = 40.5 mpg

Ah, sorry, my mistake. But getting 40.5 MPG on a freeway run shouldn't be too hard, honestly. For me I can 45+ MPG with 4 miles on the freeway. With more miles I can bump that 45 up to 50 easily, maybe 60 if I slowed down (I go 60-70 DWL, 55 is too slow for people around here).