Polymorphism validation is an important issue in genetic studies because only polymorphic markers provide useful information. We analyzed genetic data for 180 SNPs in the human major histocompatibility complex region in Caucasian and Taiwanese populations, and evaluated ethnic heterogeneity between these populations to illustrate the importance of polymorphism validation. An initial individual genotyping experiment (IGE) with 95 samples was compared with a DNA pooling allele-typing experiment (PAE) of 630 individuals for polymorphism validation based on authentic data sets. Afterwards, all samples were genotyped individually in a confirmation study. Under narrow (broad) polymorphism criteria, 24 (41) polymorphic SNPs in Caucasians could not be validated in the Taiwanese population, suggesting a 13% (23%) inconsistency rate and revealing a strong discrepancy between genetic backgrounds, probably due to ethnic heterogeneity. IGE yielded high sensitivity and specificity for polymorphism validation, but may be sensitive to sampling variation. PAE showed high sensitivity (97%) and specificity (100%) using a narrow polymorphism criterion, but reduced specificity (83%) using a broad criterion. Public domain polymorphism databases should therefore be used with caution and polymorphism validation should be performed routinely prior to conducting large-scale genetic studies. PAE is a cost-saving, reliable alternative to IGE for polymorphism validation, especially for a stringent polymorphism criterion.