Localization of homogalacturonan (HG) and xyloglucan epitopes in developing and mature pit membranes from different pit types in xylem of Populus tremula L. × P. tremuloides Michx. (hybrid aspen) and Populus tremula L. (European aspen) was investigated using immunogold labeling. Pit types not mediated by ray parenchyma (intervessel- and fiber pits) showed significant developmental changes in HG epitope localization. Both low- and high methyl-esterified HG epitopes (recognized by LM19 and LM20, respectively) were detected in developing pit membranes of intervessel- and fiber pits until late stages of xylem formation, whereas no HG- and high methyl-esterified HG epitopes were detected in mature intervessel (except for annulus regions of pit membranes)- and mature fiber pit membranes, respectively. In contrast, no notable developmental changes in HG epitope localization were detected in pit types mediated by ray parenchyma (vessel-ray-, ray- and fiber-ray pits) during pit maturation. Vesselray- and fiber-ray pits showed abundant low- and high methyl-esterified HG epitopes in pit membranes, while ray pits showed presence of primarily low methyl-esterified HG epitope during all stages of pit development including at maturity. With xyloglucan (recognized by LM15), specific developmental changes in epitope localization were detected in vessel-ray pits. Xyloglucan epitope was detected in developing vessel-ray pit membranes, but was almost absent in mature pit membranes. Instead, xyloglucan was detected in the protective layers of vessel-ray pits showing completely different localization pattern than homogalacturonan, which was only detected in pit membranes. Together, our results suggest that the chemistry of pit membranes varies depending on both the developmental stage and pit type.