Few existing studies have dealt with cytogenetics in trematodes, largely due to the attendant technical difficulty of chromosome preparation. We performed a comparative analysis of chromosomes in five opistorchiid species, including Opisthorchis felineus Rivolta, 1884, Opisthorchis viverrini Poirier, 1886, Clonorchis sinensis Cobbold, 1875, Metorchis xanthosomus Creplin 1846, and Metorchis bilis (Braun, 1790) Odening, 1962. For some of these species, no detailed morphometric description of their karyotypes has yet been published; for the karyotype of Metorchis bilis this is the first-ever description. We found that opisthorchiids, like other trematodes, are characterized by karyotypic conservatism (N = 6–7) and karyotype asymmetry, although comparison of chromosome morphometric traits did reveal differences between the karyotypes of the species. Moreover, to address certain a methodological issue in trematode chromosome preparation, we analyzed how the source of chromosomal material (partenitae or mature flukes) and the chromosome preparation techniques used (air-drying and cell suspension methods) affected chromosome spreading and size, concluding that the most reliable comparative method involves comparing relative parameters (relative length, arm ratio, centromeric index) of chromosomes prepared using the same technique.