Failure in accurate segregation of chromosomes during meiosis causes aneuploidy, the main contributor to infertility, spontaneous abortions, and developmental defects. A molecular understanding of the factors that regulate chromosome dynamics during meiosis is necessary to better understand the cellular origins of these aneuploidy events. Polo-like kinase 1 (PLK1) participates in processes during the cell cycle, such as DNA replication, mitotic entry, chromosome segregation and cytokinesis. In this study, we focused our attention on the role of PLK1 during a DNA damage repair event, homologous recombination. Previous studies have shown that PLK1 has a central role in regulating an early recombination intermediate, RAD51, as well as in influencing Class II MUS81-EME1 crossover pathway. We used a germ-cell-specific conditional knockout strategy, driven by Spo11-Cre recombinase, to assess the role of PLK1 in regulating the transition from prophase to metaphase in mouse spermatocytes. Our results showed Plk1 conditional knockout mice were infertile and displayed severe meiotic aberrancies, including enlarged primary spermatocytes and increased apoptotic cells. Interestingly, we observed alterations in recombination protein foci numbers occurring during homologous recombination. These results underscore the importance in maintaining strict temporal control of PLK1 activity during spermatogenesis