Genetic variations of three populations(Liangzi Lake population of 19, Longwo Lake population of 19 and Huaihe River population of 18) of Plagiognathops microlepis were studied by sequence analysis of mitochondrial cytochrome b(Cyt b) gene.Gene fragments of 1149 bp were acquired by PCR and DNA sequencing techniques.There were 10 haplotypes, 116 nucleotide variable sites and 10.09% of nucleotide polymorphic loci among three populations of Plagiognathops microlepis.As a base compo-sition, the content of A+T(about 57%) was obviously higher than that of C+G.Highest nucleotide diversity was observed in Huaihe River population(5 haplotypes, 111 nucleotide variable sites and 0.031223 of nucleotide diversity index), and lowest nucleotide diversity was detected in Longwo Lake population(3 haplotypes, 2 nucleotide variable sites and 0.000550 of nucleotide diversity index).Genetic distances between populations of Liangzi Lake and Longwo Lake, between populations of Huaihe River and Liangzi Lake, and between populations of Huaihe River and Longwo Lake were 0.001530, 0.084682 and 0.084335, respec-tively.There was a closer genetic relationship between populations of Liangzi Lake and Longwo Lake than that between populations of Huaihe River and the other two.Genetic difference between populations of Liangzi Lake and Longwo Lake has not risen to sub-species level, while genetic divergence between populations Huaihe River and Liangzi Lake and between populations Huaihe River and Longwo Lake may have arrived at subspecies level.