The gastrointestinal microbiota of chickens plays a central role in health and performance. Cloacal swabs, due to their proximity to the ceca (a vital site of functional activity), are an alternative, non-invasive method used for assaying microbial communities and might be a viable option for longitudinal studies. In this study, the microbiota of twenty paired cecal content and cloacal swab samples representing two dietary treatments was assessed using 16S rRNA V4 hypervariable region sequencing and was analyzed using the MOTHUR pipeline, Phyloseq, and Vegan packages. Paired t-test and Wilcoxon signed-rank tests showed significant differences in the Chao1 index (p-value <0.0001 and p-value <0.0001, respectively) but not in the Inverse Simpson species diversity estimator (p-value = 0.06763 and p-value = 0.06021, respectively) between the cecal content and cloacal swabs. β-diversity between the cloacal swabs and cecal microbiota also showed significant differences using PERMANOVA, HOMOVA, and weighted UniFrac testing (p-values < 0.001). Based on a paired sample analysis, this study provided evidence of the high inter-individual variation and randomness of cloacal microbiota, in contrast to cecal microbiota. Our findings indicated that cloacal swabs do not approximate the α or β diversity of cecal samples and are not suitable for longitudinal studies of gut microbiota.