With the decrease of cost in genotyping, single nucleotide polymorphisms (SNPs) have gained wide acceptance because of their abundance, even distribution throughout the maize (Zea mays L.) genome, and suitability for high-throughput analysis. In this study, a maize 55 K SNP array with improved genome coverage for molecular breeding was developed on an Affymetrix® Axiom® platform with 55,229 SNPs evenly distributed across the genome, including 22,278 exonic and 19,425 intronic SNPs. This array contains 451 markers that are associated with 368 known genes and two traits of agronomic importance (drought tolerance and kernel oil biosynthesis), 4067 markers that are not covered by the current reference genome, 734 markers that are differentiated significantly between heterotic groups, and 132 markers that are tags for important transgenic events. To evaluate the performance of 55 K array, we genotyped 593 inbred lines with diverse genetic backgrounds. Compared with the widely-used Illumina® MaizeSNP50 BeadChip, our 55 K array has lower missing and heterozygous rates and more SNPs with lower minor allele frequency (MAF) in tropical maize, facilitating in-depth dissection of rare but possibly valuable variation in tropical germplasm resources. Population structure and genetic diversity analysis revealed that this 55 K array is also quite efficient in resolving heterotic groups and performing fine fingerprinting of germplasm. Therefore, this maize 55 K SNP array is a potentially powerful tool for germplasm evaluation (including germplasm fingerprinting, genetic diversity analysis, and heterotic grouping), marker-assisted breeding, and primary quantitative trait loci (QTL) mapping and genome-wide association study (GWAS) for both tropical and temperate maize.