Abstract: Objective: The objective of this study was to determine the effect of different dimethylacetamide (DMA) levels on sperm quality of Bangkok rooster chicken and sperm survivability in reproductive tract of hen. Materials and Methods: Sperm was collected from 4 Bangkok rooster chicken aged 1 year. Thirty native hens were used for insemination. Sperm from roosters was collected once a week and cryopreserved 24 h in liquid nitrogen containers at -196°C using 3% (P1), 5% (P2), 10% (P3), 14% (P4) and 18% (P5) DMA levels. Frozen thawed sperm quality was observed after thawing at 4° for 60 sec. The hens were slaughtered and sperm viability was recorded in their reproductive tract on 3, 7, 14 and 21 days after insemination. Results: The results showed that freezing decreased the sperm motility, viability and increased abnormalities. The P2 was observed to be the best DMA concentration for chicken sperm cryopreservation. Different DMA concentrations significantly (p<0.01) affected the motility, viability and abnormalities of frozen thawed sperm. Sperm motility (%) after thawing in P1, P2, P3, P4 and P5 were 38.00±9.08, 46.00±8.94, 16.00±5.48, 8.00±2.24 and 1.00±2.74, respectively. Sperm viability (%) after thawing was 40.10±7.21, 53.50±12.53, 22.00±4.43, 15.30±11.40 and 12.00±3.98 in P1, P2, P3, P4 and P5, respectively. Sperm abnormalities (%) after thawing in P1, P2, P3, P4 and P5 were 60.40±7.40, 41.00±3.32, 67.20±4.09, 68.20±8.58 and 71.00±11.64, respectively. In P2, spermatozoa from the hen’s reproductive tract (vagina, uterus, infundibulum and fimbria) were found motile up to 21 days after insemination. Conclusion: It can be concluded that the best DMA concentration for chicken sperm cryopreservation was 5% level in terms of sperm quality and its survivability in the reproductive tract of hen.