We attempted to simultaneously analyze single nucleotide variations (SNVs) in a number of samples by integrating the high fidelity of ligation of universal probes with the robustness of three-dimensional (3D) polyacrylamide gel DNA microarray. By performing a ligation reaction between the 5' phosphate terminus of the sequencing primer and the 3' hydroxyl terminus of the labeled probe, we accurately identified a single nucleotide polymorphism (SNP) in the polymerase chain reaction (PCR) products of 33 genomic DNA samples and two point mutations in the PCR-amplified 83 mitochondrial DNA (mtDNA) samples immobilized in gel. Fluorescent imaging allowed genotyping with a high call rate (100%). The average fluorescence intensity obtained by ligation of universal probes was about 85% of that acquired by dual-color fluorescence hybridization, and the detection specificities of these two methods were similar. Because the fluorescently labeled probes can be used to detect all SNVs, ligation of universal probes was thought to be more cost effective. Our study demonstrated that this method can detect SNVs in an economic, authentic, and high-throughput format on the 3D microarray platform.