Utilizing formalin-fixed paraffin-embedded (FFPE) archival tissue, the most common form of tissue preservation in routine practice, for cytogenetic analysis using microarray comparative genomic hybridization (aCGH) remains challenging. We searched for a predictive factor of the performance of FFPE DNA in aCGH analysis. DNA was extracted from 63 FFPE archival tissue samples of various tissue types (31 breast cancers, 24 lung cancers, and 8 thyroid tumors), followed by aCGH analysis using high-density oligonucleotide microarrays. Tumor DNA from matched frozen samples and from FFPE samples after whole-genome amplification were also analyzed in 2 and 4 case, respectively. The derivative log ratio spread (DLRSpread) was used to assess the overall quality of each aCGH result. The DLRSpread correlated significantly with the double-stranded DNA ratio of tumor DNA, storage time, and the degree of labeling with Cy5 (P<0.0001; correlation coefficients=−0.796, 0.551, −0.481, respectively). Stepwise multiple linear regression analysis revealed that the double-stranded DNA ratio of tumor DNA is the most significant predictive factor of DLRSpread (regression coefficient=−0.4798; P=<0.0001). The cytogenetic profiles of FFPE and matched frozen samples showed good concordance. Although the double-stranded DNA ratios were increased after whole-genome amplification, the DLRSpread was not improved. The double-stranded DNA ratio can be used to predict the performance of aCGH analysis for DNA from FFPE samples. Using this quality metric, valuable FFPE archival tissue samples can be utilized for aCGH analysis.