The highly pathogenic avian influenza virus of subtype H5N1 that caused serious outbreaks in Egypt in 2006 was efficiently detected using a commercially available real-time reverse transcriptase–PCR (RRT-PCR) for the type A specific matrix (M) gene in field samples of cloacal and tracheal swabs. RRT-PCR was also used for subtyping and confirmation of H5 subtype. During late 2007 the National Laboratory for Veterinary Quality Control on Poultry Production detected five field cases that were positive for avian influenza virus (AIV) based on the M gene RRT-PCR. Three different commercial H5 RRT-PCRs were used for identification of the H5 subtype, as well as a published World Organization for Animal Health (OIE) H5 RRT-PCR that had been previously carefully validated. The five cases had positive results for the H5 gene using the published OIE H5 RRT-PCR, but the three commercial H5 RRT-PCRs tests only returned two to four positive results out of the five positive cases. The hemagglutinin gene (HA) sequencing analysis of these five isolates showed multiple nucleotide substitution mutations, suggesting genetic variation that could affect the H5 primer and/or probe binding sequences. These data highlight the importance of continued monitoring of RRT-PCR primers and probes to ensure that sensitivity and specificity are maintained. The use of conventional methods in national and reference AIV laboratories, including virus isolation, serologic subtyping, and alternative RRT-PCR primers, is necessary to detect the newly emerging variant H5N1 strains that affect diagnostic performance.