Abstract Background Schistosoma japonicum remains a major challenge to human and animal health. Earlier microsatellite-based studies reported possible definitive-host-specific private alleles within S. japonicum, opening the possibility that different definitive hosts might harbour different parasite strains. Previous investigations have also detected near-identical multilocus genotypes in populations of adult worms - possibly the result of mutations occurring during the asexual (intramolluscan) phase of clonal expansion. Research has also revealed extensive deviations from Hardy-Weinberg Proportions (HWP) and conflicting results among studies. The present study was performed to examine some of the potential effects of infrapopulation structure on microsatellite-based studies of the transmission ecology of S. japonicum. Potential sources of bias considered included organotropic distribution of worms, non-random mating and corrections for clonal expansion. Results Stool samples from naturally infected hosts were used to infect snails in the laboratory and thereby expose mice. 274 individual worms were typed at seven microsatellite loci. Removal of individuals bearing duplicate MLGs (as a correction for presumed clonal expansion) had an impact on both HWP and organotropic genetic differentiation. The study found no evidence that heterozygote deficiencies were caused by a Wahlund effect. Female-male pairings appeared to be random and there was no evidence for mate choice by heterozygosity. There was some indication that excess heterozygosity, induced by clonal expansion, can offset heterozygote deficiencies caused by small population size or populations fragmented by parasite control efforts. Conclusions The view is supported that miracidia are preferable to adult worms in investigations into host-specific parasite lineages. Where adults must be used, extreme care should be taken with regard to sampling if infrapopulations of small animals are compared with those of larger animals; this is because of organotropic patterns in genetic variation and the tendency to sample from different organs in differently sized hosts. As corrections for clones may accentuate signals of population subdivision, corrections should only be made if tests for clonal expansion prove positive. Finally, evidence for heterozygote deficiency caused by small sample size, calls for carefully designed random and comprehensive sampling strategies for S. japonicum in China, where control efforts have greatly fragmented parasite populations.