Background: Human intestinal schistosomiasis caused by Schistosoma mansoni and urinary schistosomiasis caused by Schistosoma haematobium are endemic in Ethiopia. Although schistosomes look morphologically uniform, there is variation in infectivity, egg productivity and virulence due to variation in their genetic make. Knowing the genetic diversity and population structure of S. mansoni isolates will enable to understand and consider the possible variability in terms of infectivity, egg productivity and virulence. Methods: Between 2010 and 2011, genetic diversity and population structure of Schistosoma mansoni isolates from four endemic areas of Ethiopia was assessed using previously published 11 polymorphic microsatellite loci. Miracidia were hatched from eggs of S. mansoni collected from stools of human subjects residing in Kemissie, Wondo Genet, Ziway and Sille-Elgo villages. DNA was extracted from single miracidium and PCR was run following standard protocol. Allelic polymorphism and population genetic structure was analyzed using different software. Result: At a population level (i.e. different villages), the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy–Weinberg equilibrium and pairwise F ST values ranged from 8.5 to 11.5, 3.46–20.8, 0.66–0.73 and 3.57–13.63 %, respectively. All analyzes on population genetic structure reveals strong genetic structuration corresponding to the four sampled villages. At infrapopulation level (i.e. different hosts) the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy–Weinberg equilibrium and F IS values ranged from 3.09 to 7.55, 1–1.96, 0.59–0.73 and 0.1763–0.4989, respectively. Mean estimated genetically unique adult worm pairs within hosts ranged from 66 to 92 % revealing the occurrence of infection of a single host with genetically unique multiple S. mansoni strains. The data also indicated the occurrence of genetic variation within inter-and intra-hosts. Conclusion: High level of genetic diversity and significant population differentiation characterized the S. mansoni isolates of Ethiopia. These results are quite different from previous studies demonstrating that it is difficult to generalize schistosome transmission patterns because epidemiological situation tends to vary. These are important factors to be considered in relation with morbidity, drug resistance or vaccine development.