It's not clear when a new specie is different from the previous one, but you probably need:
* Like 100.000 years, probably more. (From the split of us from chimps 5.000.000 I counted like 30 species in https://en.wikipedia.org/wiki/Human_evolution , but the number depends a lot on who count them.)
* Isolate the populations so they can't interbreed (first because they can't meet and later becuse the dna is incompatible)