Genome-wide association mapping for complex traits in cattle populations is a powerful, but expensive, selection tool. The DNA pooling technique can potentially reduce the cost of genome-wide association studies. However, in DNA pooling design, the additional variance generated by pooling-specific errors must be taken into account. Therefore, this study aimed to investigate factors such as: (i) the accuracy of allele frequency estimation; (ii) the magnitude of errors in pooling construction and in the array; and (iii) the effect of the number of replicate arrays on P-values estimated by a genome-wide association study. Results showed that the Illumina correction method is the most effective method to correct the allele frequency estimation; pooling errors, especially array variance, should be taken into account in DNA pooling design; and the risk of a type I error can be reduced by using at least two replicate arrays. These results indicate the practical capability and cost-effectiveness of pool-based genome-wide association studies using the BovineSNP50 array in a cattle population.