A 2.3 kilobase (kb) Salmonella probe, JEO402‐1, and two subfragments, F1214 (1.3 kb) and F1217 (0.8 kb), have been evaluated by colony hybridization using pure cultures of Salmonella serovars and non‐salmonella bacteria. JEO402‐1, and its subfragments, F1214 and F1217, hybridized to all of 156 different Salmonella serovars tested, while there was no reaction to 112 non‐salmonella strains belonging to 19 genera and 37 species of Enterobacteriaceae. Together with previously published results, the JEO402‐1 probe has now been shown to detect a total of 396 Salmonella strains belonging to 214 serovars of Slamonella subspecies I‐VI. A total of 178 non‐salmonella strains representing 23 genera and 51 species of Enterobacteriaceae have all tested negative with JEO402‐1. The hybridization results obtained using a digoxigenin‐labeled probe were similar to those obtained with ³⁵S isotopic labeling when complete colony lysis was ensured.