The thermal tolerance of Bemisia tabaci MED, an invasive whitefly species with worldwide distribution, plays an important role in its ecological adaptation during the invasion process. Heat-shock proteins (HSPs) are closely related to heat resistance. In this study, 33 Hsps (BtaHsps) were identified based on sequenced genome of B. tabaci MED belonging to six HSP families, among which 22 Hsps were newly identified. The secondary structures of a further 22 BtaHsps were also predicted. The results of RT-qPCR showed that heat shock could affect the expression of 14 of the 22 Hsps newly identified in this study. Among them, the expression level of six Hsps increased under 42 °C treatment. As the unstudied gene, BtaHsp90A3 had the highest increase rate. Therefore, BtaHsp90A3 was chosen for the RNAi test, and silencing BtaHsp90A3 by RNAi decreased the survival rate of adult B. tabaci at 42 °C. The results indicated that only a few Hsps were involved in the thermal tolerance of host whitefly although many Hsps would response under heat stress. This study conducted a more in-depth and comprehensive identification that demonstrates the evolutionary relationship of BtaHsps and illustrates the response of BtaHsps under the influence of thermal stress in B. tabaci MED.