Karyotypes and reproductive isolation were studied in two allopatric populations of Drosophila tsigana, one from Guizhou Province in southern China and the other from Hokkaido in northern Japan, and in one population of a closely related species, D. longiserrata, from Guizhou. In metaphase plates of larval brain cells, both geographic strains of Drosophila tsigana showed 2n=10 chromosomes, with 2 pairs of metacentric (V-shape), 2 pairs of acrocentric (R-shape), and 1 pair of dot-liked (D-shape) chromosomes. Drosophila longiserrata showed the same number, 10 chromosomes, comprising 2V, 1J (sub-metacentric chromosome), 1R, and 1D. X chromosomes of both species were acrocentric, the presumed ancestral form. Premating isolation was complete between D. tsigana and D. longiserrata, and successful mating was also limited in crosses between the two geographic populations of D. tsigana, especially in crosses between Japanese (JP) females and Guizhou (GZ) males. F1 hybrids were obtained only from crosses between GZ females and JP males, and fertilities of both F1 females and males were quite incomplete. The results of morphological observations, karyotypic analyses, and crossing experiments clearly showed that the GZ and JP populations of “D. tsigana” were highly divergent from each other and that each population should be recognized as a biologically valid species. The present morphological observations and chromosomal analyses, together with the original descriptions, strongly suggest that “Guizhou D. tsigana” might be conspecific with D. bisetata Toda, 1988 from Myanmar, and that D. longiserrata might be conspecific with D. afer Tan, Hsu, and Sheng, 1949 from Meitan, Guizhou.