Abstract Venomous animals use venom; a complex biofluid composed of unique mixtures of proteins and peptides, to act on vital systems of the prey or predator. In bees, venom is solely used for defense against predators. However, the venom composition of bumble bees ( Bombus sp. ) is largely unknown. Thoracobombus subgenus of Bombus sp. is a diverse subgenus represented by 14 members across Turkey. In this study, we sought out to proteomically characterize the venom of five Thoracobombus species by using bottom-up proteomic techniques. We have obtained two-dimensional polyacrylamide gel (2D-PAGE) images of each venom sample. We have subsequently identified the protein spots by using matrix assisted laser desorption ionization / time of flight mass spectrometry (MALDI-TOF MS). We have identified 47 proteins for Bombus humilis ; 32 for B. pascuorum , 60 for B. ruderarius ; 39 for B. sylvarum and 35 for B. zonatus . Our analyses provide the primary proteomic characterization of five bumble bee species’ venom composition.