The Bemisia tabaci cryptic species complex harbors a diversified flora of primary and secondary endosymbionts, which plays crucial roles in many aspects of the insect biology. The endosymbiont infection pattern is dependent upon many factors, including host plant and geographic origin. In Brazil, the invasion of B. tabaci Middle East-Asia Minor-1 (MEAM 1) populations was observed in tomato (Solanum lycopersicum L.) fields in the 1990s, which was followed by severe begomovirus epidemics. Here we confirmed the vertical transmission of "Candidatus Portiera" and the localization of secondary endosymbionts in distinct B. tabaci developmental stages. Hamiltonella defensa was detected in bacteriocytes but also scattered in the leg muscles as well as in the male heads. Wolbachia-specific signals were found in eggs, nymphs, male adults, and female gonads in predominant association with bacteriocytes. These results were somewhat surprising since Wolbachia infection is rarely found in MEAM 1 populations from the Old World. Our results reinforce the notion that endosymbiont infection pattern may vary according to the insect population, gender, developmental stage, and geographic origin. This characterization will provide tools to study the endosymbiont function in the transmission ability/efficiency of a complex of tomato-infecting bipartite begomoviruses by B. tabaci under Neotropical conditions.