This study investigated genetic variability within the 'eyeworm' Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by polymerase chain reaction (PCR)-coupled sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1). Eight different sequence variants of cox1 (haplotypes) were determined for the 50 individual adult specimens of T. callipaeda (from dogs, foxes or cats from Italy, Germany and the Netherlands and from dogs from China and Korea). Nucleotide variation (0.3-2%) was detected at 23 of 649 positions in the cox1. Six of these positions were invariable among all 37 individuals from Europe and among the 13 individuals from Asia (irrespective of host origin) but differed (five G'A and one C'T changes) between Europe and Asia. PCR-based single-strand conformation polymorphism (SSCP) analysis of the most variable portion (v-cox1) of the cox1 was validated (for a subset of samples) as a tool to rapidly screen for genetic (haplotypic) variability. The results for the SSCP analysis and sequencing were concordant, indicating that the mutation scanning approach provides a useful tool for investigating the population genetics and molecular ecology of T. callipaeda.