Analysis of allelic loss in archival tumor specimens using array technology is constrained by the quality and quantity of available tissue. A prototype Affymetrix HuSNP array was shown to provide reliable and reproducible assessment of allelic imbalance for 440 SNPs in frozen esophageal tumors (Mei-2000). However, the commercially-available Affymetrix HuSNP array (1,494 SNPs) has not been validated for the assessment of allelic imbalance in tumors processed by standard pathology methods. We tested the HuSNP assay in duplicate on breast specimens using both formalin-fixed and frozen, tumor and normal tissue taken from a single patient (16 arrays). Tumor cells were purified using bivariate cytokeratin/DNA flow sorting; normal breast served as constitutive normal. STR typing on 3 chromosomes validated regions of allelic imbalance. Allele calls from the HuSNP array averaged 95% reproducibility between duplicates and 94% concordance between the fixed and frozen samples. We also tested DNA from the same samples that was subjected to whole genome amplification (Primer Extension Pre-amplification, PEP) prior to array analysis. Although overall signal intensities were lower, the data from the PEPed material was reproducible in duplicates and concordant between sample types at similar rates to genomic DNA. Results from genomic normal tissue DNA averaged informative (AB) calls at 379 loci over all chromosomes. Although data points were clustered and large segments of chromosomes were not informative by this technique, our data indicated that the Affymetrix HuSNP assay could potentially provide a low resolution genome-wide analysis of allelic imbalance in routinely processed pathology specimens.