The whitefly species Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) are worldwide agricultural pests and virus vectors. Bemisia tabaci, in particular, is often transported internationally via trade routes leading to potential introductions of exotic whiteflies or plant viruses. Quick identification of agriculturally important whiteflies can facilitate interventions that prevent these cross-border introductions. Polymerase chain reaction (PCR) primers were designed to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) sequence of members of the B. tabaci complex, MEAM1, MED, and NW, and T. vaporariorum. Primers incorporated an A/T-rich overhang sequence at the 5′ terminus (5′ flap) to test for increased primer sensitivity and assay efficiency. Single-target and multiplex endpoint PCR assays with the eight primer sets were performed using genomic DNA template extracted from individual adult whiteflies. Resultant PCR amplicons obtained for B. tabaci MEAM1, MED, and NW, and T. vaporariorum primers with the 5′ flap were 559-, 717-, 353-, and 258-bp, respectively, and without the 5′ flap were 550-, 712-, 329-, and 252-bp in length, respectively. In single-target and multiplex reactions, specific amplification was achieved using both the unmodified and 5′ flap-modified primers. Sequencing and phylogenetic analysis confirmed primer-target amplification specificity. Using these primer sets in single-target or multiplex PCR allows for quick discrimination and specific identification of B. tabaci complex members and T. vaporariorum, and the addition of 5′A/T-rich overhang sequences increases the sensitivity and amplification of some primer sets.