The recently released Affymetrix GeneChip® Medicago Genome Array contains approximately 52 700 probe sets representing genes in both the model legume Medicago truncatula Gaertn. and the closely related crop species Medicago sativa L. (alfalfa). We evaluated the utility of the Medicago GeneChip® for monitoring genome-wide expression of M. truncatula and alfalfa seedlings grown to the first trifoliate leaf stage. We found that approximately 40–54% of the Medicago probes were detected in leaf or root samples of alfalfa or M. truncatula. Approximately 45–59% of the detected Medicago probes were called ‘present’ in all replicate GeneChips of Medicago species, indicating a considerable overlap in the number and type of Medicago probes detected between root and leaf organs. Nevertheless, gene expression differences between roots and leaf organs accounted for approximately 17% of the total variation, regardless of the Medicago species from which the samples were harvested. The result shows that the Medicago GeneChip® is applicable for transcript analysis for both alfalfa and M. truncatula.