Type I acyl-CoA: diacylglycerol acyltransferase (DGAT1) is a rate-limiting enzyme that catalyses the final step of the Kennedy pathway in triacylglycerol biosynthesis. In this study, two genes encoding type I diacylglycerol acyltransferase, ZmDGAT1a and ZmDGAT1b, were cloned from a high-oil maize inbred line and confirmed by Southern blotting. The complete coding sequences were obtained. Comparison between genomic DNA and coding sequences revealed that both ZmDGAT1a and ZmDGAT1b comprised 16 exons and 15 introns with a coding region of 1485 and 1488 bp, respectively. ZmDGAT1a and ZmDGAT1b shared similar molecular mass, isoelectric point, and transmembrane domains. ZmDGAT1b was expressed in all sampled tissues that were examined by using reverse transcription-polymerase chain reaction (RT-PCR). In contrast, ZmDGAT1a expression was limited to the silks, tassels, and seeds of different ages. This suggests that the two genes do not have a complementary relationship. The ZmDGAT1a and ZmDGAT1b proteins were subsequently found to be localized in the endoplasmic reticulum (ER). The data provides an important basis for further studies of the functions of DGAT1 genes and for genetic modification of the oil content and composition of corn seeds.