The rumen flukes of 37 cows (Bos indicus) from Chiang Mai and Lamphun provinces were investigated, and the overall prevalence of infection was 78.38% (29/37). Three species were found: Paramphistomum epiclitum, Orthocoelium streptocoelium, and Fischoederius elongatus with prevalences of infection of 75.68%, 48.65%, and 40.54%, respectively. Genomic DNA was amplified by polymerase chain reaction based on the high annealing temperature-random amplification of polymorphic DNA (HAT-RAPD) technique. Five random 10-mer oligonucleotide primers (OPA2, OPA4, OPB18, OPC9, and OPH11) produced distinct banding patterns in three species. No genetic variations in these three species were identified using 10 arbitary primers.