The Mediterranean group (commonly known as Q biotype; hereafter MED) of the sweetpotato whitefly, Bemisia tabaci (Gennadius), originated in the Mediterranean region, but it now has been found in at least 10 countries outside the Mediterranean. Collections of B. tabaci from some of these countries exhibit different pest behaviors and pesticide resistance characteristics, yet all may be classified as MED. A phylogenetic analysis of 120 mitochondrial cytochrome oxidase I (mtCOI) sequences (JN966761–JN966880) of MED whiteflies collected in Arizona and of 417 retrieved from the GenBank database resolves the MED into five subclades, designated as Q1–Q5. Only subclades Q1 and Q2 have been detected in the United States. Q1 and the other four subclades (Q2–Q5) differ in the number or position of the AluI recognition sites. Based on the differences in the AluI recognition sites reported here and the previously reported differences in VspI recognition sites, we developed a simple diagnostic technique to identify subclades Q1–Q5 by using mtCOI polymerase chain reaction (PCR) -restriction fragment-length polymorphism (RFLP). A test of a worldwide collection of whiteflies demonstrates that this combination mtCOI PCR-RFLP technique can reliably distinguish not only the MED from the Middle East-Asia Minor 1 group but also the Q1 from any of the other four MED subclades.