Enterobacterial isolates producing CTX-M β-lactamases have recently emerged worldwide in the community and hospital settings. Because of the significant public health implications, the spread of organisms producing CTX-M enzymes merits close monitoring with enhanced surveillance efforts. A molecular diagnostic assay using two different sets of primers simultaneously for the detection of all blaCTX-M-like β-lactamase genes was developed. This assay repeatedly demonstrated 100% sensitivity, specificity and positive and negative predictive values for detecting different CTX-M enzymes in well-characterised strains that included producers of VEB-, TEM- and SHV-type extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC enzymes. The majority (132/240; 55%) of ESBL-producing enterobacterial isolates recovered in the Calgary Health Region during 2003 and 2004 were positive for blaCTX-M genes, including 81 (61%) positive for the CTX-M-9 group, 49 (37%) for the CTX-M-1 group, and two (2%) for the CTX-M-2 group. The CTX-M-specific PCR assay was reproducible and easy to use. It can be introduced in a clinical or reference laboratory to track and monitor the spread of organisms producing CTX-M enzymes in the community and hospital settings.