Herein, we propose rapid and simple spectroscopic methods to determine the chemical composition of propolis derived from various Populus species using a metabolomics approach. In order to correlate variability in Populus type propolis composition with the altitude of its collection, NMR, IR, and UV spectroscopy followed by OPLS was conducted. The botanical origin of propolis was established by comparing propolis spectral data to those of buds of various Populus species. An O2PLS method was utilized to integrate two blocks of data. According to OPLS and O2PLS, the major compounds in propolis samples, collected from temperate continental climate above 500 m, were phenolic glycerides originating from P. tremula buds. Flavonoids were predominant in propolis samples collected below 400 m, originating from P. nigra and P. x euramericana buds. Samples collected at 400–500 m were of mixed origin, with variable amounts of all detected metabolites.