Potato cyst nematodes cost the potato industry substantial financial losses annually. Through the use of molecular markers, the distribution and infestation routes of these nematodes can be better elucidated, permitting the development of more effective preventative methods. Here we assess the ability of three molecular markers to resolve multiple representatives of five Globodera pallida populations as monophyletic groups. Molecular markers included a region of the rbp-1 gene (an effector), a noncoding nuclear DNA region (the ITS region), and a novel marker for G. pallida, a ;3.4 kb non-coding mitochondrial DNA (mtDNA) region. Multiple phylogenetic analysis methods were performed on the three DNA regions separately, and on a data set of these three regions combined. The analyses of the combined data set were similar to that of the sole mtDNA marker; resolving more populations as monophyletic groups, relative to that of the ITS region and rbp-1 gene region. This suggests that individual markers may be inadequate for distinguishing populations of G. pallida. The use of this new non-coding mtDNA marker may provide further insights into the historical distribution of G. pallida, as well as enable the development of more sensitive diagnostic methods.