Brain-derived neurotrophic factor ( BDNF) has multiple alternative splicing variants and plays diverse biological functions in mammals, including neuronal survival, cholesterol metabolism, cell differentiation and tumor development. However, genomic structures of chicken BDNF (c BDNF) variants and its potential functions are still undefined. Here, we characterized two novel alternative splicing variants of c BDNF, c BDNF1 and c BDNF2, via combining comparative genomics methods and molecular techniques in inbred chicken line 6 3 and line 7 2, which have been developed to be resistant and susceptible, respectively, to Marek's disease tumor since 1939. Both c BDNFs consist of a bipartite transcript, with different 5′ exons, exon I (298 bp) in c BDNF1 and exon II (286 bp) in c BDNF2, each of which is spliced to the common 3′ exon IV. Exon I and IV are highly conserved between chicken and mammals, whereas exon II is unique for chicken. The amino acid sequence of cBDNF1 contains 8 additional amino acids in the N terminal compared to cBDNF2. c BDNF1 and c BDNF2 were only expressed in the hypothalamus among eight tissues, and c BDNF2 showed lower expression than that of c BDNF1 in both lines. The expression level of c BDNF1 was significantly higher in line 7 2 than in line 6 3 ( P < 0.01). Notably, the DNA methylation levels on the cis-regulatory region of c BDNF1 was negatively correlated with its expression level, which suggests that the mRNA expression level of c BDNF1 may be related to the DNA methylation status in the chickens. We also discussed a potential role of variant c BDNF1 in MD tumor resistance and susceptibility.