The five species in the genus Esox are popular gamefish that form a unique phylogenetic group based on their characteristic duckbill morphology as well as recent molecular data. To better understand the inter-specific relationships in this genus, the beta-actin gene was isolated from each of the five species, and sequence comparisons were carried out for nucleotide and amino acid positions within the gene. Additionally, an internal region of each actin gene family member was sequenced from Esox masquinongy. In contrast to published reports on Takifugu rubripes, where nine actin genes were identified, only six actin genes were detected in the esocids. The similarity and differences between these family members is outlined and the evolution of the actins is discussed. The significant intronic divergence between the actin family members among the five Esox species makes multi-actin gene amplification by polymerase chain reaction (PCR) a potential diagnostic marker for species within a genus, and suggests its use in hybridization or population studies as an alternative to other DNA fingerprinting techniques.