This study aimed to characterize the lentogenic NDV isolate from native chickens in Surabaya, Indonesia. Thirty seven samples of cloacal swabs from infected native chickens were collected, and pathotypes were characterized using Mean Death Time (MDT) analysis; six isolates were found to be lentogenic. Lentogenic NDV isolates were then analyzed via RT-PCR, using primers specific for cleavage site of fusion protein (F). PCR product was sequenced and analyzed using Epitope Prediction Tools (IEDB) Analysis Resource to determine the epitopes. The results showed some shifts in nucleotides, but no change was observed in amino acids. Five samples were found with similar sequence in the cleavage site, except NDV isolated from sample Ck/sby27, which had a different amino acid sequence, “RRQKRFI.” Epitope characterization specific for T-cell NDV was found in Ck/sby27 at position 42-142, with highest real score in epitopes CLDYLQLVY, SIDGRPLAA, and TAEQITAAA. These findings reinforce the assumption that the original NDV lentogenic strain is of wild origin and is still circulating in the environment. Therefore, NDV isolate Ck/ sby27 can be used in developing vaccines in the future.