Background: Poonch is the remotest district of Jammu, India located near to line of control with Pakistan. Poonchi chicken is the first native poultry breed of Jammu region which is being characterized and registered with National Bureau of Animal Genetic Resources. Present study is the first study on genetic bottleneck assessment in Poonchi chicken population using recommended microsatellite markers. This study in Poonchi chicken would be of immense use in tracing evolutionary origin and formulating effective conservation strategies for genetic diversity within breeds. Methods: Blood samples were collected randomly from Poonch and Rajouri districts of Jammu for DNA extraction and analysis. Microsatellite markers selected for the analysis were ADL0268, MCW0206, MCW0081, ADL0278, MCW0069, MCW0248, MCW0111, MCW0222, MCW0016 and LEI0094. To evaluate the Poonchi chicken for mutation drift equilibrium, Bottleneck software was used. Infinite allele model (IAM), two phase model of mutation (TPM) and stepwise mutation model (SMM) tests were done. Mode shift test was also used as another method of qualitative test for allele frequency. Result: Total number of alleles observed were 72, with the total number alleles ranged from 6 to 9. The results of all three models (IAM, TPM, SMM) in Sign test depicted the heterozygosity was more in the expected number of loci in Poonchi chicken as 5.97 (P less than 0.05), 5.93 (P less than 0.05), 5.90 (P less than 0.05) respectively. Whereas for Wilcoxon rank test the IAM, TPM and SMM revealed significant values of (P less than 0.05) deviation indicating that all the loci deviate from mutation-drift equilibrium. Mode shift test graph obtained depicted a slight shift from normal L-shaped curve indicating recent bottleneck effect in Poonchi chicken. This might have resulted in the loss of number of effective alleles which suggests a need of planning and implementing an effective breeding policy in order to preserve and promote this native breed.