Females have nipples. Males do not. This is easily seen on their lower bellies.
Their genital to anal distance is also different. Females have a very short distance between the vaginal opening and anal opening. The male has a further distance between the penis and anus. Not to mention his scrotal sac will drop at around 4-5 weeks of age. So you could always just wait for that to happen before splitting the litter.
Around 3 weeks of age.
Female:
Male:
5 week olds
Female:
Male:
Those pictures are from this forum. It shows a pretty good guide of how to sex rats.
http://australianratforum.com/forum/...ustrated-Guide
http://www.ratz.co.uk/sexingrats.html
http://www.ratforum.com/showthread.p...xing-Baby-Rats