In this study, we use restriction fragment length polymorphisms (RFLP) of the mtDNA ND2 gene to identify to species cyprinid eggs collected from a spawning aggregation substrate in the Pascagoula River system. A library was generated with inter- and intra-specific RFLP patterns for nine cyprinids: Notropis baileyi, N. texanus, N. longirostris, N. amplamala, Nocomis leptocephalus, Luxilus chrysocephalus, Lythrurus roseipinnis, and Cyprinella venusta. Eggs of three species were identified from a single nest, while only one species was observed spawning at the site. These results confirm that RFLP is an effective method for identifying unknown eggs of nest associates, and North American cyprinids in general.