Diacylglycerol acyltransferase 1 (DGAT1) gene has become a potential candidate gene for milk composition and yield in cattle. A nonconservative lysine to alanine substitution (K232A) at position 10.433 and 10.434 in the eighth exon of DGAT1 gene has widely studied in cattle, goat, and sheep. This substitution conducted a considerable effect on milk fat, which was previously mapped to the centromeric end of bovine chromosome 14. Yet, no information has been found about DGAT1 gene in donkeys. In this study, the genetic variation of DGAT1 gene has been analyzed by CfrI (EaeI) restriction and DNA sequencing in 85 donkeys reared in Thrace region of Turkey. No digestion is found in the DGAT1 gene via CfrI restriction enzyme in the studied donkey populations. The DNA sequence of 437 bp of the DGAT1 gene in donkeys revealed only Alanine allele (K232A), which was reported in cattle with high milk fat content. But we found a novel polymorphism at position 10.435, from G to A, which also revealed Alanine allele. As a conclusion, CfrI digestion is not a diagnostic site in DGAT1 gene in donkeys. The DNA sequence of DGAT1 gene in donkeys is reported for the first time in this study, and this sequence is deposited to National Center for Biotechnology Information GenBank database with the accession number MK086162. (C) 2018 Elsevier Inc. All rights reserved.