R2R3-MYB transcription factors play important role in transcriptional controls during higher plant metabolism and development. In this study, an R2R3-MYB gene was isolated from maize according to an EST, which expressed differentially between a hybrid and the two parents on a cDNA chip. The full-length cDNA, designated by ZmMYBL1 (GenBank accession no. AY365033) consists of 1417 nucleotides and contains an open reading frame of 828 bp. The deduced amino acid sequence contained two conserved MYB domains near its N-terminus, a conserved E1 motif and an acidic Ser/Thr rich region toward its C-terminus. Southern blot analysis revealed ZmMYBL1 could be a single copy gene belonging to a multi-gene family in the maize genome. Expression analysis showed ZmMYBL1 transcripts accumulated in various tissues examined, with strong level in tassel and weak level in leaf. Also it was under-expressed in root, stem, and leaf of hybrid as compared with that of the two parents. ZmMYBL1 was mapped on maize chromosome bin7.03 between two SSR markers, bn1g339 and umc1865 using Yuyu22 recombinant inbred line population. A QTL for root average diameter in maize seedlings was also localized on the corresponding region of chromosome 7 within the interval ZmMYBLI-bnIg1805. A possible role of ZmMYBL1 and its relation to maize heterosis were discussed based on these results.