Parasitic nematodes of the genus Haemonchus infect a range of ruminant hosts and are of major veterinary and economic importance. In this study, the genetic variability of seven isolates of Haemonchus placei and Haemonchus contortus was evaluated using the mitochondrial gene cytochrome oxidase subunit I and the nuclear gene b-tubulin isotype 1. A total of 156 specimens were obtained from cattle, sheep, goat and buffalo herds raised on commercial properties from the southern and southeastern regions of Brazil and identified to the species level by sequencing of the nuclear internal transcribed spacer 2. Thirty-four percent of the specimens were identified as H. placei and 66% as H. contortus. Cattle were the preferred hosts for H. placei, whereas H. contortus was most frequent in the other three ruminant species. Analysis of genetic differentiation between isolates revealed that high rates of gene flow are operating among populations of both nematode species, including among those from different ruminant host species. Populations of H. placei were less polymorphic and presented a lower frequency of single nucleotide polymorphisms associated with benzimidazole resistance compared with H. contortus. In line with the low amount of genetic structure observed among isolates, alleles of b-tubulin 1 associated with benzimidazole resistance were present at relatively high frequencies of 5­20% in isolates of H. contortus from farms that never used this class of anthelmintic. The results presented here are consistent with the hypothesis of multiple origins of alleles associated with benzimidazole resistance, with the trade of animals among properties acting as the main factor promoting the spread of anthelmintic resistance.