Atlantic (Acipenser oxyrinchus oxyrinchus) and shortnose sturgeon (Acipenser brevirostrum) are broadly distributed along the Atlantic Coast of North America, where they use rivers, estuaries, and coastal habitats. In order to support management under the U.S. Endangered Species Act, it is important to understand when and where these fish occur. However, this presents a challenge as the two taxa are sometimes misidentified and some life stages (e.g., eggs) are challenging to identify with confidence. In this study, we used cytochrome b sequences to develop molecular primers to confirm the identity of shortnose and Atlantic sturgeon samples. We tested these primers using reference DNA samples for these two species. The results suggested that the primers were able to positively identify and distinguish Atlantic and shortnose sturgeon. The accuracy of the Atlantic sturgeon primers was 95.34%, whereas the accuracy of the shortnose sturgeon primers was 90.7%. Even though there were some individuals that were not positively identified as their corresponding species (false negatives), we did not observe any false positives. Our paper does not aim to develop eDNA markers; rather, the objective of our study was to create species-specific, unlabeled, and cost-effective primers which can be amplified using conventional PCR. The amplification product can be observed in a 2% agarose gel run through electrophoresis. This entire procedure is relatively inexpensive and involves basic instruments found in most conservation genetics laboratories.