As a kind of binding protein, the type 1 Na(+)/H(+) exchanger (NHE1) is a receptor for the highly pathogenic Avian leukosis viruses-J subgroup (ALV-J) in chicken. In order to investigate the potential effect of chicken NHE1 gene on leukosis, we compared its expression between ALV-J-affected and -unaffected chicken, screened variations across the whole gene, and then performed association analysis with ALV-J affected/unaffected trait in three un-related chicken populations. We found that the NHE1 gene expressed in four immune tissues including spleen, bursa fabricius, liver, and thymus, and its expression was significantly up-regulated in liver and thymus of ALV-J-affected chickens (with leukosis phenotype) compared to -unaffected ones (ALV-J-negative controls). Thirty-six single nucleotide polymorphisms (SNP) were identified in a 6,105 bp region of the chicken NHE1 gene, giving rise to every 170 bp per SNP. Two SNP of g.4405A>G and g.5886C>G were genotyped with PCR-RFLP method. Results showed that g.4405A>G was significantly associated (P < 0.05) with ALV-J infection in all of the three chicken populations, including White Recessive Rock (WRR), Dwarf Yellow (DY) and Shiki Yellow (SY), while g.5886C>G was significantly associated (P < 0.05) with ALV-J infection in SY. These results indicated that the NHE1 gene was related to ALV-J infection in chicken.