Using different genetic lines of pepper, this study analyses the effect of boron (B) on pollen quality and its capacity to pollinate and fertilize. Three different B concentrations (100, 150, and 200 mg L−1) were applied to six genetic lines, evaluating the in vitro pollen quality and pollen formation. In a second experiment, the highest and the lowest B concentrations were applied to two low-pollen genetic lines, evaluating the growth of the pollen tubes in flowers pollinated with treated pollen. The higher B concentrations significantly improved pollen quality and formation in vitro, while improved pollination and fecundity was observed after monitoring the pollen tubes. For all variables, the specific genetic line also showed some effect. This technique represents a tool for improving pollen in manual hybrid seed programs.