Non-lethal sampling techniques were used to document the reproductive structure of the shortnose sturgeon (Acipenser brevirostrum) population at a fish aggregation site (near Bordentown, New Jersey) in the Delaware River. A total of 68 fish were captured using gill nets (100 × 1.8 m, 12.7 or 15.2 cm mesh) and examined laparoscopically in May–June, and 61 additional fish captured and examined in November during 2006–2011. Six stages of reproductive development were identified in females and five stages were identified in males, encompassing differentiation through maturity in both sexes. Fish captured in the spring were predominantly immature with a higher proportion of females (1 : 1.2 M : F sex ratio), while mature males predominated in the autumn (5.7 : 1 M : F), indicating that the Bordentown area serves as an overwintering/pre-spawn aggregation site. Three distinct forms of intersex were noted in gonads of 11.6% of fish examined: ovo-testis, consisting of scattered spermatic cysts in predominantly immature ovary (3.9%); testis-ova, consisting of ovarian lamellae projecting from a predominantly immature testes (4.7%); and zonal distribution, consisting of multiple, nearly-homogenous pockets of either testicular or ovarian tissues along the gonad (3%). The hormone profile in fish with ovo-testis was similar to that of immature males, while the hormone profile in fish with testis-ova was similar to that of immature females. The relatively high prevalence of intersex raises concerns regarding potential reproductive effects and long-term impacts on shortnose sturgeon in the Delaware River.