Sex and reproductive maturity of Atlantic and shortnose sturgeon were determined by visual examination of the gonads using laparoscopy, and were validated by histological examination of gonadal biopsies. Surgical anesthesia was induced in all fish with 250 mg L−1 tricaine methanesulfonate (MS-222) and maintained throughout procedures with 85 mg L−1 MS-222 on a mobile surgical cart. A pair of Ternamian EndoTip cannulae installed through the ventral body wall in each fish, allowed access for a 5-mm rigid laparoscope and biopsy forceps. Video endocamera use with the laparoscope, following air insufflation of the coelom, provided detailed, high quality imagery to aspirate the swim bladder, examine the gonad and collect biopsies without inducing iatrogenic trauma. Germinal tissue of all immature males, 25% of immature female shortnose sturgeon and 45% of immature female Atlantic sturgeon were concealed by fat preventing sex determination by visual assessment. Morphological features of gonads were used to determine sex in all remaining fish and were 100% in concordance with histological findings. Relative amount of gonadal fat; gonad size and color; presence of testicular lobes or ovarian lamellae; and color, size and density of oocytes were useful in determining reproductive stage. Gonad morphology of each reproductive stage was similar in Atlantic and shortnose sturgeon. All captive Atlantic sturgeon survived laparoscopy, gained weight at the same rate as unexamined fish and scars from incisions were no longer evident 9–12 months after surgery. Laparoscopic procedures presented here offer a safe and highly reliable way to determine sex and reproductive status for Atlantic and shortnose sturgeon.