A subtracted library was constructed of genes expressed specifically in the chick precardiac mesoendoderm. The subtracted library was obtained by hybridization of nucleic acids derived from a starting tester library of stage 4–7 chick precardiac mesoendoderm and a starting driver library of stage 2 area pellucida. Approximately 11,000 clones from the resulting subtracted library were printed onto a microarray. Screening of the microarray with probes derived from cardiac and noncardiac tissues, followed by in situ hybridization during chick embryo development, has identified multiple cardiac-specific genes, including several that have not been characterized previously. The microarray will be useful for future attempts to identify additional novel cardiac-specific genes, as well as to characterize patterns of gene expression during heart differentiation. Developmental Dynamics 230:290–298, 2004. © 2004 Wiley-Liss, Inc.