Deletion of 9p21 is the most commonly reported chromosomal abnormality in pediatric acute lymphoblastic leukemia, and published data suggest that the maternal chromosome is preferentially deleted. Preferential maternal deletion of 9p21 and reports of a differentially methylated region (DMR) and of parental effects in mice with lymphoma suggest there may be an unrecognized imprinted locus in this region. To screen for DMRs, we used the mcrBC/HpaII screening method and peripheral-blood DNA. Of 36 CpG islands within an 8.5-Mb region of 9p21, seven were identified as putative DMRs and were further analyzed by bisulfite sequencing. Neither any of the CpG islands nor a previously published putative DMR nearby showed evidence of differential parental methylation; however, the published DMR did demonstrate sequence-dependent differential methylation. Our data, which showed heterogeneous and low-level methylation of CpG islands, have obvious implications for methylation studies.