The potato cyst nematode (PCN), Globodera pallida, originates from South America and is considered one of the most severe agricultural pests of potato crops and other Solanaceae plants globally. Based on their virulence and ability to reproduce on various potato cultivars, the populations of G. pallida are divided into three pathotypes, Pa1– Pa3. In this study, comparative sequence analyses of the fragment of mitochondrial cytochrome c oxidase subunit II (mtCOII) gene for eight populations of G. pallida, representing three pathotypes, Pa1, Pa2 and Pa3, indicated genetic diversity between them. However, we did not identify significant mutations distinguishing Pa2 from Pa3. Interestingly, two single nucleotide substitutions, T441C and A468G, were characteristic only for populations assigned to Pa1. On this basis, we developed high resolution melting (HRM) PCR protocol. As a result, the melting curves obtained for samples of Pa1 populations varied from those obtained for populations designed as Pa2 and Pa3, allowing their differentiation. Thus, the HRM protocol developed here enables a rapid, very sensitive and low-cost screening assay for SNPs identification in mtCOII of G. pallida pathotypes. In effect, it might also be a helpful molecular tool in pathotype differentiation. However, further verification of the correlation of the occurrence of single nucleotide mutations in mtCOII in particular pathotypes should be carried out on a much larger number of samples of G. pallida, to determine if these mutations are characteristic only for this pathotype.