The genetic structure of populations of the fish cestode, Bothriocephalus acheilognathi collected from Bailianhe Reservoir (BLH), Changshou (CSH) and Liangzi (LZH) Lakes was investigated by using 8 microsatellite loci. A total of 108 adult worms were genotyped at each of the 8 loci. For the 3 populations, the mean number of alleles per locus ranged from 2·38 to 5·5, and the mean expected heterozygosity ranged from 0·432 to 0·559. The average polymorphic information content (PIC) was from 0·384 to 0·492. The significant Fis values indicated non-random mating within LZH and BLH populations. On the other hand, when samples were further classified into subpopulations at the level of host fish species, no or little heterozygote deficiency was detected at most loci, showing that cross-fertilization, predominantly, but not exclusively, must have occurred within the subpopulations. Microsatellite markers also revealed an unexpected high level of genetic differentiation, as measured by Rst and Nm values or by δu2 genetic distance among subpopulations from different hosts. Factors influencing the population genetic structure and the parasite host specificity are discussed.