Color is not a distinguishing characteristic that can differentiate the two Gambusia. Like you said, Fishoreel, color can be largely influenced by habitat. If you really want to know the difference, you need to look at the gonopodium on the males. The gonopudium ray has a series of 'teeth' on it in holbrooki, and is smooth on affinis. You'll need magnification to see this. There are dorsal fin ray counts too, but in all honesty, I wouldn't bother...