Mildew resistance locus O (MLO) is a plant-specific gene family that plays an important role in the growth and development of plants and their interactions with the environment.  However, the available information on this gene family in pear is limited.  Here, 24 PbrMLO genes were identified and divided into five subfamilies (I, II, III, IV and V).  Whole-genome duplication (WGD) and dispersed duplication contributed to the expansion of the PbrMLO family.  In addition, gene expression analysis revealed that PbrMLO genes were distributed in various pear tissues, suggesting their diverse functions.  We selected PbrMLO23 for further functional analysis.  Expression profile analysis by qRT-PCR showed that PbrMLO23 was highly expressed in pollen.  Subcellular localization analysis showed that PbrMLO23 was located on the plasma membrane.  When the expression level of PbrMLO23 was knocked down by using antisense oligonucleotides, pollen tube lengths increased, indicating that PbrMLO23 plays a functional role in inhibiting pollen tube growth.  In summary, these results provide evolutionary insight into PbrMLO and its functional characteristics and lay a foundation for further analysis of the functions of PbrMLO members in pear.