The reproductive performance (e.g. fertility) of dairy cows, which declined over past few decades due to the intense and intensive selection, needs to be improved. Previous genome-wide association study (GWAS) of female Holstein screened the Adenylate cyclase 5 (ADCY5) as the candidate gene for cow fertility. As a member of the adenylyl cyclases family, adenylate cyclase 5 (ADCY5) is famous for regulating extrapyramidal motor system related various neuropsychiatric diseases, and its genetic variant is reported to associate with lower birth and placenta weight which leads to asymmetric fetal growth restriction. It was hypothesized that ADCY5 may affect the fertility of cows by regulating the processes of ovarian development. Herein, genomic DNA from 768 ovaries samples of healthy unrelated Holstein cow in dry period were used to screen potential insertion/deletion (indel) mutations using eight pairs of primers, and we found three novel polymorphic indel variants, namely, rs385624978 (P3-D), rs433028962 (P5-I) and rs382393457 (P8-D). The minor allelic frequencies (MAF) of P3-D, P5-I and P8-D loci were 0.188, 0.365 and 0.06, respectively, and there were 7 different haplotypes. Additionally, linkage disequilibrium analysis demonstrated no linkage among them. Importantly, P3-D locus was significantly related to both ovarian width (P=1.0E-6) and corpus luteum diameter (P=0.015); P5-I locus had a significant relation with corpus albicans diameter (P=0.030) and ovaries with mutational homozygous genotype produced a superior corpus albicans diameter than those with other genotypes. Briefly, three novel indel mutations of bovine ADCY5 gene were identified and two of them were uncovered to be significantly correlated with ovarian phenotypic traits or corpus luteum or albicans traits. These findings contributed to the application of molecular marker-assisted selection (MAS) in improving female fertility in cattle, which could accelerate the development of the cattle industry.Copyright © 2020 Elsevier B.V. All rights reserved.