The sweetpotato whitefly, Bemisia tabaci (Gennadius), is an important pest worldwide. A new biotype of sweetpotato whitefly, biotype B, causes damage by direct feeding and by the transmission of plant viruses, such as geminiviruses. In the Mediterranean area, tomato yellow leaf curl geminivirus (TYLCV) is the most serious disease of tomatoes. Another whitefly-transmitted geminivirus, tomato mottle geminivirus (ToMoV), is presently a serious problem in tomato production in west-central and southwestern Florida. Because of the increasing incidence of whitefly-transmitted geminiviruses, it is necessary to develop rapid and simple diagnostic methods for the detection of viruliferous whiteflies. The polymerase chain reaction is a sensitive and specific technique for the detection and identification of plant pathogens. Polymerase chain reaction methods were used successfully to amplify 1.1-kb DNA fragments from individual viruliferous B. tabaci carrying either TYLCV or ToMoV, and no amplified DNA fragments were obtained when nonviruliferous B. tabaci adults were processed similarly. Southern hybridization analysis proved that fragments amplified from viruliferous B. tabaci adults were viral DNA. This polymerase chain reaction-based detection method is sensitive enough to detect TYLCV and ToMoV in individual viruliferous B. tabaci in mixed samples of up to 25 (1 viruliferous: 24 nonviruliferous) and 10 (1 viruliferous: 9 nonviruliferous) individuals, respectively. The potential uses of this polymerase chain reaction-based detection method in epidemiological studies of geminiviruses are discussed.