The current definition of coliform bacteria is method-dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy in identifying true coliforms. This study used an alternative approach to identify true coliforms by combing the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences together with the use of lacZ and uidA genes. A collection of 1404 isolates from 12 US Environmental Protection Agency approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation medium and Lauryl Tryptose broth, m-Endo and MI agar media. Isolates were phylogenetically classified into 32 true coliform or targeted Enterobacteriaceae (TE) groups, and 14 non-coliform or non-targeted Enterbacteriaceae (NTE) groups. It was statistically shown that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I, Providencia, Proteus, and Morganella), and most false-positive (FP) events with two NTE groups, Aeromonas and Plesiomonas. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by those enzymatic methods were validated as FNs. The reasons behind the FP and FN reactions could be explained through the analysis of the lacZ and uidA gene. Overall, combining the analyses of 16S rRNA, lacZ and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods.