The whitefly Bemisia tabaci MEAM1 is a notorious vector capable of transmitting many plant viruses, resulting in serious crop loss and food shortage around the world. To investigate potential sRNA-mediated regulatory mechanisms in whiteflies that are affected by virus acquisition and transmission, we conducted small RNA (sRNA) deep sequencing and performed genome-wide profiling of piwi-interacting RNAs (piRNAs) in whiteflies that were fed on tomato yellow leaf curl virus (TYLCV)-infected or non-infected tomato plants for 24, 48, and 72 h. In the present study, piRNA reads ranging from 564,395 to 1,715,652 per library were identified and shown to distribute unevenly in clusters (57 to 96 per library) on the whitefly (B. tabaci MEAM1) genome. Among them, 53 piRNA clusters were common for all treatments. Comparative analysis between libraries generated from viruliferous and non-viruliferous whiteflies identified five TYLCV-induced and 24 TYLCV-suppressed piRNA clusters. Approximately 62% of piRNAs were derived from non-coding sequences including intergenic regions, introns, and untranslated regions (UTRs). The remaining 38% were derived from coding sequences (CDS) or repeat elements. Interestingly, six protein coding genes were targeted by the TYLCV-induced piRNAs. We identified a large number of piRNAs that were distributed in clusters across the whitefly genome, with 60% being derived from non-coding regions. Comparative analysis revealed that feeding on a virus-infected host caused induction and suppression of only a small number of piRNA clusters in whiteflies. Although piRNAs primarily regulate the activity of transposable elements, our results suggest that they may have additional functions in regulating protein coding genes and in insect-virus interactions.