A membrane feeding system and polymerase chain reaction (PCR) were used to track squash leaf curl virus (SLCV) DNA in whole whitefly body extracts and in saliva, honeydew, and hemolymph of its whitefly vector, Bemisia tabaci, and a whitefly nonvector, Trialeurodes vaporariorum. SLCV ingestion was monitored by PCR in whiteflies that were given acquisition access periods (AAPs) ranging from 0.5 to 96 h on virus-infected plants. SLCV detection by PCR in whole body extracts was considered reflective of virus ingestion. As whiteflies were given longer AAPs, the number of whiteflies that ingested SLCV increased. SLCV DNA was detected in honeydew of vector and nonvector whiteflies, indicating that virions, viral DNA, or both passed unimpeded through the digestive system. SLCV DNA was detected in saliva and hemolymph of B. tabaci, but not in these fractions from nonvector whiteflies, despite virus ingestion by both. Although vector and nonvector whiteflies both ingested SLCV, only in the vector, B. tabaci, did virus cross the gut barrier, enter the hemolymph, or pass into the salivary system. These results suggest that digestive epithelia of nonvector whiteflies did not permit SLCV passage from the gut to hemocoel, whereas virus effectively crossed the analogous gut barrier in vector whiteflies.