cDNA microarray spot variability arises from many sources, and different systems have varying requirements for achieving the desired level of precision. We determined relative contributions to variance and investigated sequence discrimination using a multiple-array experimental design, with arrays subdivided to determine position and pin effect. Related fragments of 67 resistance gene homologs (RGHs) isolated from Theobroma cacao L. and grouped by sequence similarity were spotted onto arrays, using two of the same RGHs in the fluorescent dye channels (Cy3, Cy5) of the hybridization solution in a "dye-flip" design. A comprehensive statistical model accounted for variability well, giving a coefficient of variation (CV) based on experimental error of 2.12%. Although we were able to separate 85% of RGH group means clearly, some groups more similar to the target were indistinguishable due to nonspecific hybridization. Genetic factors together contributed 72.2% of the total variation, while position and pin effects and their interactions contributed 9.8%. Replication effect was statistically significant. Otherwise, no tests for position effects were significant. The results of the analysis indicate that our Genetic Microsystems 417 arrayer and Affymetrix 428 scanner are performing with sufficient precision, and we produced useful information for planning efficient future experiments.