Background: Resistance to β lactamase by Escherichia coli is one of the principal molecular mechanisms leading to failure of extended spectrum cephalosporin antibiotics. Nevertheless, family-based PCR results are often considered evidence of ESBL phenotype despite the non-ESBL parent gene identified by sequencing. Objectives: Analysis of clinical isolates collected from Iraq using a molecular genetic approach considering alleles, including antimicrobial susceptibility testing, multiplex PCR, partial gene sequencing, phylogenetic analysis and ERIC PCR fingerprinting. Methods: Fifty two isolates were characterized by conventional identification, disk diffusion, phenotypic ESBL and AmpC tests, multiplex PCR targeting bla TEM, bla SHV, bla CTX M, bla OXA, and six plasmid mediated AmpC families, partial Sanger sequencing, and ERIC PCR. Data were analyzed using Wilson 95% confidence intervals, Cohen's κ, and exact McNemar tests. Results: The highest resistance frequencies were observed for tetracycline (67.3%), trimethoprim/sulfamethoxazole (55.8%), and nalidixic acid (42.3%); 40.4% of isolates met the definition of multidrug resistance (MDR; resistance to ≥3 antimicrobial classes). Thirty five isolates (67.3%) carried at least one screened β lactamase family marker. blaTEM was detected in 29 isolates (55.8%), blaOXA in eight (15.4%), blaSHV in one (1.9%), blaDHA in three (5.8%), and blaCTX M in none. Sequencing assigned the detected alleles to TEM 1, SHV 1, OXA 1, and DHA 1. Consequently, the molecular results demonstrate β lactamase gene carriage but do not justify labeling all PCR positive isolates as ESBL producers. Phenotype–marker concordance was poor for the ESBL comparison (42.3%; κ = −0.03; p = 0.0014) and limited for AmpC (71.2%; κ = 0.025; p = 0.0074). ERIC PCR resolved 16 profiles among 35 gene positive isolates at 50% similarity, indicating substantial clonal heterogeneity. Conclusion: Allele discrimination essentially alters the way family level PCR results should be analyzed. In order to properly classify resistance and perform surveillance in an Iraqi medical setting, both current phenotypic criteria and allele discrimination sequencing must be used.