Genes associated with sex differentiation (the process by which undifferentiated gonads develop into testes or ovaries) are relatively conserved among vertebrates, but those associated with sex determination (the genetic and/or environmental “switch” that activates differentiation) are more variable, particularly in fishes. Virtually nothing is known regarding the genetic basis of these processes in lampreys, one of the two extant groups of jawless fishes. Therefore, I tested whether 19 candidate sex determination genes identified from other vertebrates showed sex specific sequence differences in sea lamprey, Petromyzon marinus, and Pacific lamprey, Entosphenus tridentatus; 11 and seven genes amplified in sea and Pacific lampreys, respectively, but none showed sex specific differences. I also used qRT-PCR to measure expression of seven candidate sex differentiation genes (e.g., SOX9, DMRT1, WT1, DAZAP1) prior to and during testicular differentiation in sea lamprey, and found that expression patterns were consistent with their presumed role in other vertebrates.