AbstractThe oomyceteAphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogenA. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studiedA. astacistrains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating fromOrconectessp.,Pacifastacus leniusculusandAstacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from theP. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating fromProcambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from theOrconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of theA. astacihaplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.