Quantitative evidence on tree space segregation among avian assemblages in temperate primeval forests is currently lacking. Therefore, our study aimed to determine the species composition of birds across different parts of trees. We investigated whether species richness differs between positions on a tree and how these positions influence the probability of occurrence of specific bird species. To achieve this, we observed birds within permanent plots in Białowieża National Park (BNP) and analyzed the distribution patterns of birds within six vertical and three horizontal sectors of trees. The compositional dissimilarity between tree sectors was assessed using detrended correspondence analysis. We employed generalized linear mixed-effects models to examine differences in species richness and the probability of occurrence for ten of the most frequently observed species across tree sectors. The majority of the BNP bird community was associated with the branches, while other birds occupied the tree crown trunks and the understory trunks. Species richness was the highest on branches in the crown part of trees, followed by lower species richness on trunks associated with crowns, and the lowest richness was observed on branches and trunks in the understory. These results indicate that branches in the middle and lower parts of the crown serve as avian diversity hotspots on trees, likely due to the abundance of various food sources. The differing patterns of tree usage by specific bird species may suggest the avoidance of interspecific competition for resources. Importantly, our study provides a pristine state for future investigations.