Hyperpigmented melanistic skin lesions (HPMLs) of smallmouth bass Micropterus dolomieu are observed in the Potomac and Susquehanna rivers, Chesapeake Bay watershed, USA. Routine, nonlethal population surveys were conducted at 8 sites on the mainstem Susquehanna River and 9 on the Juniata River, a tributary of the Susquehanna River, between 2012 and 2018, and the prevalence of HPMLs was documented. A total of 4078 smallmouth bass were collected from the mainstem Susquehanna River and 6478 from the Juniata River. Lesions were primarily seen in bass greater than 200 mm, and prevalence in the Susquehanna River (8%) was higher (p < 0.001) than in the Juniata River (2%). As part of ongoing fish health monitoring projects, smallmouth bass were collected at additional sites, primarily tributaries of the Susquehanna (n = 758) and Potomac (n = 545) rivers between 2013 and 2018. Prevalence in the Susquehanna River (13%) was higher (p < 0.001) than the Potomac (3%). Microscopically, HPMLs were characterized by an increased number of melanocytes in the epidermis or within the dermis and epidermis. RNAseq analyses of normal and melanistic skin identified 3 unique sequences in HPMLs. Two were unidentified and the third was a viral helicase (E1). Transcript abundance in 16 normal skin samples and 16 HPMLs showed upregulation of genes associated with melanogenesis and cell proliferation in HPMLs. The E1 transcript was detected in 12 of the 16 melanistic areas but in no samples from normal skin. Further research will be necessary to identify the putative new virus and determine its role in melanocyte proliferation.