Abstract: In order to identify MHC haplotype of chicken embryo fibroblasts（DF-1 cell）, DF-1 cells with good growth status were selected in the experiment to extract total RNA, and the total RNA was reverse transcribed into cDNA. Using DF-1 cell cDNA as a template, the microsatellites LEI0258 and MCW0371 of chicken MHCB locus and BF2、BLB2 genes were amplified and sequenced using PCR method; BF2 gene was amplified and sequenced using the “PCR+1” method simultaneoustly, and determine the MHC haplotype of DF-1 cells based on above sequences and their structural characteristics. The results showed that the length of microsatellite LEI0258 and MCW0371 sequences were 357 bp and 205 bp, respectively, and microsatellite LEI0258 contained one CTATGTCTTCTTT repeating Unit and 16 CTTTCCTTCTTT repeating Units, which were exactly the same as the microsatellite length, repeating unit structure, and quantity of MHC-B locus B130, B131, B201, B5.1, B6.1, B21, and B75 haplotypes. The length of the BF21 gene sequence was 1 117 bp, which was completely consistent with the BF2 sequences（registration numbers AF013493.1, S78682.1, NM-001031338.2） from all three B21 haplotype sources included in GenBank. The results indicated that DF-1 cells were derived from B21 haplotype homozygous chicken embryos.