We evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4-V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1-V3(1) and (2), and V3-V4 and V3-V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.