Sex determination of fish species is difficult to assess when sexual dimorphism and gametes are not apparent. For threatened and endangered fish species, noninvasive techniques are needed when determining sex to minimize stress and the potential for mortality. We evaluated the use of a portable ultrasound unit to determine sex of Lake Sturgeon Acipenser fulvescens in the field. Ultrasound images were collected from 9 yellow-egg (F2, F3), 32 black-egg (F4, F5), and 107 fully developed male (M2) Lake Sturgeon. Two readers accurately assigned sex to 88–96% of fish, but accuracy varied in relation to maturity stage. Black-egg females and fully developed males were correctly identified for 89–100% of the fish sampled, while these two readers identified yellow-egg females only 33% and 67% of the time. Time spent collecting images ranged between 2 and 3 min once the user was comfortable with operating procedures. Discriminant analysis revealed the total length : girth ratio was a strong predictor of sex and maturity, correctly classifying 81% of black-egg females and 97% of the fully developed males. However, yellow-egg females were incorrectly classified on all occasions. This study shows the utility of using ultrasonography and a total length : girth ratio for sex determination of Lake Sturgeon in later reproductive stages around the spawning season.