Avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increasing pathogenicity and stronger horizontal and vertical transmission ability in different kinds of chicken. Although vertical transmission of ALV-J from hens infected through artificial insemination with ALV-J infected semen was reported before by the detection of swabs and serum, there was no further research on the trasmission pattern of ALVs in the roosters. In the present study, the introduction of Hy-line brown roosters infected with ALV-J significantly increased the p27 positive rate of ALV in a indigenous flock detected by virus isolation. Sequence analysis and IFA showed that it is classified into ALV-J subgroup, locating in a new branch compared with the domestic and foreign referential sequences. Meanwhile, the gp85 gene of the ALV-J isolated in the hens and its albumens had a homology of 94.1-99.7% with that in the roosters, which means that the strain is quite likely transmitted to the hens and their offspring through insemination of the roosters. Moreover, Semens are directly detected by ELISA method is not completely accurate. There are four ALV-J infection status in plasma and semen of rooster (V+S+, V-S+, V+S-, V-S-), so the eradication of ALV in rooster requires simultaneous virus isolation of semen and plasma. Therefore, we speculate that the reason why there are still some sporadic findings of ALV-J in laying hens is probably due to the incomplete eradication process of roosters.