Among the 7,220 male samples analyzed in this study, the X chromosome-specific allele at the Amelogenin locus was not amplified in 3 samples using the AmpFLSTR Profiler Plus amplification kit; however, the X chromosome-specific allele in these samples was amplified using the GenePrint Sex Identification System-Amelogenin amplification kit. The most probable explanation for this anomalous phenomenon is that these samples had a mutation of the X chromosome within the primer-binding site for the specific primer provided in the AmpFLSTR Profiler Plus amplification kit. Although this was observed only in the X chromosome in male samples, this does not preclude the possibility of a similar mutation being present in the Y chromosome or other STR locus. In forensic casework, when the sex of evidentiary samples is unknown, a careful interpretation of data is necessary if this type of anomaly is observed. In rape cases, when the presence of semen, sperm, or both is identified, a differential extraction of the sample is done to separate the sperm from epithelial cell DNA. If the female fraction is consistent with the female victim and the male fraction shows only X and no Y product, a flag would be raised suggesting no Y amplification. Such anomalous amplifications that result from mutation at the primer-binding site may be investigated by using another multiplex or singleplex system that features primers with alternative sequences. 10 references