Copy number variation (CNV) is an important type of genetic variation contributing to phenotypic differences among mammals. Up to now, GWAS analysis using CNV called by array CGH is lacking in livestock like Holstein cattle. The objectives of this work are to identify CNVs using high-density aCGH data and explore functional CNVs which are associated with complex traits by GWAS method in Holstein cattle. In this study, we reported a systematic CNV association analysis of CNVs and 39 complex production traits in Holsteins. This research identified 1043 CNV regions (CNVRs) by array CGH data in 47 Holstein bulls. Using a genome-wide association analysis (GWAS) approach, we identified 79 significant CNVRs associated with at least one complex traits after false discovery rate (FDR) correction. Notably, 24 CNVRs were markedly related to daughter pregnancy rate (DPR). This study observed the pleiotropy phenomenon of 39 CNV loci which can simultaneously regulate at least 2 complex traits. In summary, the significant CNVs identified in this research could be utilized additional molecular markers for genetic improvement programs in Holsteins. Overall design: Whole genome high-denstiy CGH arrays manufactured by NimbleGen containing 2,166,464 oligonucleotide probes were designed and fabricated on a single slide to provide an evenly distributed coverage on cattle UMD3.1 with an average interval of ~1.2 kb between probes. A total of 48 samples were analyzed. 47 of them were male Holsteins for test samples. Six of them have been uploaded in previous work. Their GSE numbers are GSM1537659, GSM1537660, GSM1537661, GSM1537662, GSM1537663, GSM1537664 in Platform GPL11314. Herein, we uploaded other 41 test samples (semen-BTHO-7~semen-BTHO-47) and one reference sample (L1 Dominette 01449, a Hereford cow of European descent and named its result as DTTRACE).