Background: Overexpression of mitotic kinases has been associated with prognosis, histologic grade, and clinical stage in ovarian cancer, but the relationship between inherited variation in these genes and ovarian cancer risk has not been well defined. Methods: We measured associations between 397 single nucleotide polymorphisms (SNPs) from 67 mitotic kinases and invasive epithelial ovarian cancer risk in two case-control studies (n = 671 cases; n = 939 controls). Thirty-six candidate SNPs (P < 0.05) were assessed in a replication analysis consisting of three additional studies (n = 1,094 cases; n = 829 controls). Results: In initial analysis, thirty-six SNPs were suggestive of association with risk of serous ovarian cancer, all subtypes of ovarian cancer, or both (P < 0.05). Replication analyses suggested an association between rs2125846 in the Nemo-like kinase (NLK) gene and ovarian cancer (serous OR = 1.36, 95% CI: 1.11-1.67, P = 1.77 × 10 -3; all subtypes OR = 1.30, 95% CI: 1.08-1.56, P = 2.97 < 10 -3). Furthermore, rs2125846 was associated with risk in the combined discovery and replication sets (serous OR = 1.33, 95% CI: 1.15-1.54; all subtypes OR = 1.27, 95% CI: 1.12-1.45). Conclusions: Variation in NLK may be associated with risk of invasive epithelial ovarian cancer. Further studies are needed to confirm and understand the biologic relationship between this mitotic kinase and ovarian cancer risk. Impact: An association between SNPs in NLK and ovarian cancer may provide biologic insight into the development of this disease. ©2012 AACR.