Host genotype and gender are among the factors that influence the composition of gut microbiota. We studied the population structure of gut microbiota in two lines of chickens maintained under the same husbandry and dietary regimes. The lines, which originated from a common founder population, had undergone 54 generations of selection for high (HW) or low (LW) 56-day body weight, and now differ by more than 10-fold in body weight at selection age. Of 190 microbiome species, 68 were affected by genotype (line), gender, and genotype by gender interactions. Fifteen of the 68 species belong to Lactobacillus. Species affected by genotype, gender, and the genotype by gender interaction, were 29, 48, and 12, respectively. Species affected by gender were 30 and 17 in the HW and LW lines, respectively. Thus, under a common diet and husbandry host quantitative genotype and gender influenced gut microbiota composite.