A new species, Crepidostomum affine n. sp., is described from Hiodon tergisus in Mississippi, and morphological data are provided for Crepidostomum auritum from Aplodinotus grunniens in Mississippi and for Crepidostomum illinoiense from Hiodon alosoides in Minnesota. The new species is most similar morphologically to C. illinoiense, but has a shorter intertesticular space, measuring 0–74 μm (mean = 19.3 ± 23.1 SD in 73 specimens) compared with 0–229 μm (mean = 57.3 ± 56.7 SD in 34 specimens), and the distance between the ovary and the anterior testis is relatively shorter in the new species, representing 2.6–7.9% of overall body length compared with 4.1–12.4% in C. illinoiense. Fragments of nuclear ribosomal as well as mitochondrial DNA are compared among C. affine n. sp., C. illinoiense, C. auritum and Crepidostomum cornutum. Crepidostomum affine n. sp. and C. illinoiense are most similar, having between 19 and 20 variable bases (1.29–1.36%) in the amplified nuclear ribosomal RNA fragment comprising the complete ITS2 spacer and partial 28S gene, and between 35 and 39 variable bases (8.62–9.61%) in the amplified fragment of the COI region. Specimens of C. illinoiense from the Missouri River in North Dakota and Red Lake River in Minnesota differed by 1 base (0.07%) in the rRNA fragment and 4 bases (0.95%) in COI fragment. Crepidostomum cornutum and C. auritum also have 19 (1.29%) variable bases in the amplified ITS2 and partial 28S regions and 50 (12.32%) variable bases in the amplified COI region. Both C. cornutum and C. auritum demonstrated much greater levels of differences compared to C. affine n. sp. These results add to previously published data reporting species of fish digeneans that might be endemic to the Pearl and Pascagoula river basins in Mississippi.