The present study was planned under the project “Development and Application of Decision Support Tools to Conserve and Sustainably Use Genetic Diversity in Indigenous Livestock and Wild Relatives” funded by UNEP-GEF-ILRI-FAnGR (United Nations Environment Programme-Global Environment Facility-International Livestock Research Institute-Farm Animal Genetic Resources) to study the production systems along with phenotypic and genetic characterization of the nine chicken populations in six randomly selected villages within two agro-ecological zones (Site-I; sandy desert and Site-II; irrigated plains) of Punjab province involving participatory rural appraisals, household surveys and in-depth monitoring tools. Information collected through Participatory Rural Appraisals showed that the main purpose of chicken keeping was home consumption and family income. Preferred chicken breeds among moderate and poor farmers were Fayoumi (FYM), Rhode Island Red (RIR) and Desi and were kept for egg production while Aseel (ASL) was the choice of well off and kept for prestige or hobby. Egg production, size of egg and plumage color was the traits preferred for FYM and RIR, broodiness, mothering ability, disease resistance and taste of meat for Desi and Naked Neck (NNK) while aggressiveness and body stature were highly ranked for ASL. Household surveys showed that most of the farm activities were headed by the household head yet women were involved in all farm activities generally and in chicken keeping particularly at both sites. More number of farmers was involved in chicken keeping at Site-I as compared to Site-II. Semi-intensive chicken production system with free range during day time and confined under shed at night with birds fed on household grains along with scavenging was reported in majority of the cases throughout the year. Average prices of birds were higher at Site-II and were highest for ASL. Health coverage was poor at both sites with most of the farmers treating birds at the help of their own or with neighbors and friends. There was almost absence of any formal training from any NGO or any government department. Sources of information about chicken related issues were poor. Chicken and eggs were marketed at farm gate, village markets or to neighbors and income obtained from extra sale of chicken and eggs was mostly kept by women, followed by household heads. Indepth monitoring revealed higher number of laying hens than other age classes among flocks. The disease was major reason for exit and bird entries were mainly through household hatched hatch and purchase. Most of the birds at both sites were multi colored or brown in plumage with undefined feather pattern. Body weights and linear measurements were higher for ASL and birds at Site-I than those at Site II. Genetic characterization of nine chicken populations viz, ASL; NNK; FYM; RIR; FRR (FYM x RIR); FRA (FYM x RIR x ASL); FRU (FYM x RIR x unknown); OLD (other local Desi) and OCB (other crossbred) using 20 microsatellite markers were found to be polymorphic. The number of alleles per locus within chicken populations ranged from 2 to 14 with an average of 9.6 alleles per locus. Structure and Principal Coordinate Analysis was clustered into two distinct groups with two maternal lineages distributed among the nine populations. These results indicated that although these nine chicken populations have already been intermixed up to some extent yet most of them were genetically different enough to be considered as separate breeds or genetic groups.