Honey bees (Apis melifera L.) collect pollen from plant anthers, mix it with salivary secretions containing various enzymes (amylase, catalase) and about 10% of nectar, place it in specific baskets on their hind legs (bee pollen), they bring it into the hive and store in the honeycomb cells. Bee pollen is the main source of protein for honey bees, and it is also used as food (categorized as a functional food or as a food supplement). The aim of the paper was to identify the major constituents and to determine the differences in overall chemical composition between the multiflora and uniflora bee pollen. Samples of bee pollen were collected using pollen traps placed on the entrance of the hives situated at three experimental locations in the Republic of Croatia: Krapina (Continental region), Otočac (Mountain region), and Senj (Adriatic region). Pooled samples (multifloral bee pollen; n = 16) were sorted by botanical origin in order to separate samples of unifloral bee pollen (n = 48). Bee pollen samples were analysed by Fourier-transform infrared spectroscopy (FTIR) coupled with ATR recording technique. The results of spectral analysis revealed that there were no significant differences in the chemical composition of multifloral bee pollen collected from different experimental locations, while the results of analysis of unifloral bee pollen showed unique spectral profiles of analysed samples despite the overlapping effects of certain absorption bands (especially molecular vibrations of proteins and carbohydrates) in particular spectral regions.