AbstractThe bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus) × blue (I. furcatus) hybrid catfish cultured in the south‐eastern United States. Emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence‐based PCR (rep‐PCR) using ERIC I and II primers was used to screen 158 E. piscicida diagnostic case isolates. A subsample of 39 E. piscicida isolates, representing predominant rep‐PCR profiles, was further characterized using BOX and (GTG)5 rep‐PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep‐PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E. piscicida representatives from each MLSA group resulted in 14‐day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p < .05), supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture in the south‐eastern United States.