Temperate maize (Zea mays L.) breeding programs often rely on limited genetic diversity, which can be expanded by incorporating exotic germplasm. The aims of this study were to perform characterization of inbred lines derived from the tropical BS39 population using different breeding methods, to identify genomic regions showing segregation distortion in lines derived by the DH process using spontaneous haploid genome doubling (SHGD), and use case-control association mapping to identify loci controlling SHGD. Four different sets were used: BS39_DH and BS39_SSD were derived from the BS39 population by DH and single-seed descendent (SSD) methods, and BS39×A427_DH and BS39×A427_SSD from the cross between BS39 and A427. A total of 663 inbred lines were genotyped. The analyses of gene diversity and genetic differentiation for the DH sets provided evidence of the presence of a SHGD locus near the centromere of chromosome 5. The case-control GWAS for the DH set also pinpointed this locus. Haplotype sharing analysis showed almost 100% exclusive contribution of the A427 genome in the same region on chromosome 5 of BS39×A427_DH, presumably due to an allele in this region affecting SHGD. This locus enables DH line production in exotic populations without colchicine or other artificial haploid genome doubling.