Biochemical changes during the germination of pine (Pinus ponderosa Laws.), and spruce (Picea pungens Engelm.) pollen grains were measured in vivo using Fourier transform infrared spectroscopy. Samples of dry, hydrated, and germinating pollen were analysed using cylindrical internal reflectance. Spectra were recorded at regular intervals up to the time of tube emergence. Germination was evaluated microscopically. The spectra revealed differences in exine structure/thickness and a significant difference in phytate content in pine and spruce pollen. Changes observed during germination included increased respiratory activity, changes in membrane lipid phase and amount, changes in protein amount and secondary structure, and changes reflecting the utilization of phytate and formation of cell walls. Infrared spectroscopy, in a short time and using a limited amount of sample, can provide biochemical insight to the process of pollen germination and has potential for the development of a more detailed analysis of pollen viability and vigor.