Drosophila lacertosa is widely distributed from northern India to Far East of Russia throughout China. We have studied geographical distributions of three kinds of chromosomal karyotypes, type D (2n=10, 4 pairs of V-shaped metacentric chromosomes and a pair of micro-chromosomes), type L (2n=10, 5V with a pair of large-sized heterochromatic autosomes) and type M (2n=10, 5V with middle-sized ones). Type D was found exclusively in local populations of D. lacertosa distributed in Yun-Gui Plateau, southwestern China. Both type L and M have a wide range of distribution, and the former occurred in subtropical regions of China including Taiwan Island, whereas the latter in cool temperate regions of East Asia covering Far East of Russia, Korea and the Japan Islands. A strong pre-mating isolation was detected between flies with type D and those with type L or M. These data demonstrate that genetic differentiation leading to cryptic speciation might have occurred in natural populations of Drosophila lacertosa.