Reproductive efficiency is arguably the most economically important trait in commercial beef cattle production, as failure to achieve pregnancy reduces the number of calves marketed per cow exposed to breeding. Identification of variation in the genome with predictive merit for reproductive success would facilitate accurate prediction of daughter pregnancy rate in sires enabling effective selection of bulls producing daughters with improved fertility. Copy number variation (CNV) is one form of structural variation that results in abnormal copies of DNA in the genome. Our lab previously reported a region between 25 and 70 Mb on chromosome 5 associated with reproductive efficiency. To further evaluate this region for genomic variations such as CNV, we initially applied a genomewide association approach based on the >700,000 SNP marker assay and PennCNV analysis to 68 Bos indicus cross females from a large commercial ranch in central Florida. A genomic segment located on chromosome 5, spanning the region of approximately 58.37 to 58.44 Mb (Bovine UMD3.1 assembly) was identified as containing a deletion associated with decreased reproductive efficiency. To verify this deletion, a quantitative real-time PCR test was developed to evaluate additional females in the central Florida and U.S. Meat Animal Research Center (USMARC) populations. The frequency of the homozygous deletion was 7% (23/319) in the central Florida population (Bos indicus cross) for females with 2 consecutive failed breeding seasons and 0% in the USMARC population (predominantly Bos taurus) of low reproductive females. To date, we have not identified the deletion in Bos taurus cattle, suggesting that the deletion is specific to Bos indicus cattle. From these data, we have identified a deletion on chromosome 5 associated with reproductive efficiency in Bos indicus-influenced cattle.