Mating strategy and insemination site varies from organism to organism. In some squids, such as those in the family Loliginidae, upon copulation, females receive male-delivered sperm capsules, spermatangia, from dimorphic sized males and store at two different body locations: the buccal membrane and the end of the oviduct. However, in lolignid kobi squid Loliolus sumatrensis, a species of Loliginidae, the females possess three insemination sites: buccal membrane (BM), basal left IV arm (ARM) and lateral head behind the left eye (EYE), therefore we studied such the unusual phenomena. First, we attempted to identify patterns of usage of these sites in relation to size differences in male individuals, and the mating history, maturity, fecundity, as well as growth indices of female individuals. Additionally, microsatellite markers were developed to explore the paternity level of site-dependent spermatangia. Unlike most other loliginid squids, no dimorphism was found in male size. The seasonal dynamics of a population in the Seto Inland Sea revealed a set priority for the initial use of insemination sites as BM, followed by ARM and then EYE, whereas the maximum number of stored spermatangia was greater in EYE>ARM>BM. Thus, the choice of site to be inseminated was not dependent on full occupancy of preferred insemination sites, but the mating history of the female. Female maturity status was correlated with the usage pattern of insemination sites but not with the number of stored spermatangia at any insemination site. We found multiple paternity at all sites. Surprisingly, few dominant males inseminated at three sites simultaneously during single mating episode. These imply that males inseminate at different sites in a female according to the mating history and maturity status of the female.