The cause of an ongoing mortality of hybrid tilapias Tilapia nilotica × T. aurea in a Texas fish farm was a biotype of Streptococcus iniae. Identification was based upon classical biochemical and physiological analysis as well as ribosomal RNA gene sequencing. The causative agent, a β-hemolytic Streptococcus species, grew better at 37°C than at 10, 25, or 40°C, and its growth was inhibited at pH 9.6, in 6.5% NaCl, and in 40% bile. The bacterium was resistant to ampicillin and furazolidone but was susceptible to several antibiotics including tetracycline, oxytetracycline, and sulfadimethoxine-ormetoprim (5:1). This is the first record of this bacterial species affecting fish in the USA. External signs of disease in tilapia were loss of orientation, exophthalmia, corneal opacity, and petechia around the mouth, anus, and proximal margins of the pectoral fins. Internally, fluid accumulation in the peritoneal cavity and enlargement of the liver, spleen, and kidney were observed.