Crossing experiments were conducted between the Naples I (NI) strain of Ophryotrocha labronica labronica, three strains of O. l. pacifica from Japan (M3), Hawaii (WH) and Los Angeles (LA), two strains of O. labronica from Australia and one from an unknown origin, discovered in a Moscow aquarium, to determine the relationships between the European and Pacific subspecies, and the newly discovered strains. Previously published data combined with new results revealed several stages in the process of speciation within the sibling group. Interpopulation crosses yielded an increased sex ratio that appears to be positively related to genetic distance and to the sex ratio of the parent strains. Fecundity data demonstrated that the three northern Pacific strains are more closely related to each other than to any other strain, confirming that they are a valid subspecies, and indicate that they are the result of natural distribution. The strain of unknown origin as well as the Australian strains clearly belong to O. l. labronica. Its isolated presence in the South Pacific may be a remnant of an earlier wider distribution or an introduction of the Mediterranean population, as the life histories of Ophryotrocha species have made them prime candidates for anthropogenic dispersals.