The neon flying squid Ommastrephes bartramii is found circumglobally in subtropical and temperate waters in the world and supports important fisheries in the North Pacific Ocean. The population of the North Pacific O. bartramii comprises a fall spawning cohort and a winter-spring spawning cohort. Most of the fall cohort occurs east of 170°E, whereas the winter-spring cohort is widely distributed across the North Pacific Ocean. Two spawning cohorts can be further subdivided into four stocks: a central and an eastern stock of the fall cohort separated near 160°W, and a western and a central-eastern stock of the winter-spring cohort separated near 170°E in which the fall cohort was the target of a driftnet fishery before 1993 while the west stock of winter-spring cohort became main fishing target of an international jigging fishery. Being a short-lived ecological opportunist, neon flying squid exhibits large fluctuations in stock abundance and distribution on the feeding grounds and large fluctuating recruitment on the spawning grounds in response to environmental variability. In order to improve our understanding of population structure, the genetic diversity of O. bartramii populations in the North Pacific Ocean was examined using microsatellite markers. Two types of biotin-labeled oligos were employed and part of microsatellite-enhanced genomic library of O. bartramii was constructed by the method of streptavidin magnetic beads. Sixty of 68 positive clones comprised microsatellite sequences and repeat number of more than 10 accounted for 86.84% with the largest one of 33. Of these were 60.53% perfect, 36.84% were imperfect, and the rest were compound type (2.63%). Repeats including (ACAG)_n and (AGAC)_n were also detected except that of AC/TG and AG/TC. Microsatellite markers were isolated and screened, and 8 of them were then used for the analysis of genetic diversity and genetic structure of six populations of O. bartramii in the North Pcific Ocean. Results showed that all the 8 loci were highly polymorphic (PIC=0.787-0.987), and locus Bo103 and locus Bo105 were extremely significant deviated from Hardy-Weinberg equilibrium (P < 0.01). All of the 6 different geographic populations showed high genetic diversities (H_o=0.672-0.761, H_e=0.808-0.851). Pairwise F test and AMOVA analysis detected no significant genetic differentiations among populations, and genetic differences mainly came from individuals. UPGMA clustering tree based on Nei's genetic distance demonstrated that two stocks (NE1 and NE3) in the northeast of the North Pcific Ocean clustered together, while the three stocks (NW1, NW2 and NW3) in the western part as well as one stock (NE2) in the eastern part clustered together, and the genetic distance between NW1 and NE2 was closer. Relations between genetic distance and geographic distance did not show positive correlation (R=0.175, P > 0.05), indicating that different populations of O. Bartramii could mingle together. Genetic structure simulation analyses suggested that there was only one logic population of all individuals, and extensive gene exchange might occur among the populations, which could be attributed to the influence of ocean currents and swimming ability of O. bartramii individuals. The North Pacific O. bartramii should be considered as a single stock in their assessment and management.