Hypotrigona species are difficult to identify morphologically. Here, we show that nest sites and nest architecture can be used to discriminate three Hypotrigona species found in Kenya. Hypotrigona gribodoi, H. araujoi and H. ruspolii colonies from Kakamega forest and H. gribodoi from Mwingi, were collected and placed in a meliponiary at the International Centre of Insect Physiology and Ecology (ICIPE). The following parameters were recorded: nest sites, internal nest entrances, external nest entrance colour and size, sizes (in terms of volume) of brood cells, honey and pollen pots, arrangement of brood cells and presence or absence of involucrum (cerumen covering brood). It was found that nest sites are specific to species. Hypotrigona gribodoi nests mostly in crevices in mud walls while H. ruspolii and H. araujoi nest in cavities in specific tree species, mainly in indigenous forests. The colour of external nest entrances varies between the species. H. araujoi’s is yellowish brown, H. gribodoi’s is white or cream while that of H. ruspolii is dark brown. There is an internal nest entrance in H. gribodoi, which is absent in the other two Hypotrigona species. Brood cells are clustered in H. gribodoi and H. ruspolii whereas H. araujo's form vertical semi comb-like layers. The area of the apical opening of the entrance tube and volumes of brood cells, honey and pollen pots differ significantly between the three Hypotrigona species. Therefore, nest sites and nest architecture can be used to discriminate three Hypotrigona species. Furthermore, the study indicates that conservation of indigenous forests, the main habitat for H. araujoi and H. ruspolii is important for their conservation.