Background The gastrointestinal microbiota in chicken ( Gallus gallus domesticus ) has a central role in health and performance. The ceca are a vital site of functional activity, but assessing cecal microbiota in longitudinal studies remains a challenge. The cecal communities are broadly similar to large intestine samples. Cloacal sampling, due to their proximity to the large intestine, is an alternative, non-invasive method used for assaying and monitoring disease-causing agents, and maybe a viable option for longitudinal studies. Results We collected paired cecal content, and cloacal swab samples from twenty randomly selected commercial broilers raised on two dietary treatments. The microbiota of each sample was assessed using 16S rRNA V4 hypervariable region sequencing on an Illumina MiSeq platform and analyzed using the MOTHUR pipeline. Analysis of fourteen paired samples resulted in 1603 OTUs assigned to 82 Families. Eleven families were shared between the cecal and cloacal samples, with seven and eleven families unique to cecal content and cloacal swabs, respectively. Paired t-test and Wilcoxon Signed-Rank test showed significant differences in the Chao1 index between the cecal content and cloacal swabs (p-value = 0.000845 and p-value = 0.001397, respectively). However, the Inverse Simpson species diversity estimator was not different using the Wilcoxon Signed-Rank test (p-value = 0.3258) and a paired t-test (p-value = 0.3864). -diversity between the cloacal swabs and cecal microbiota also showed significant differences based on PERMANOVA (p-value = <0.001), HOMOVA (p-value <0.001), and Weighted Unifrac (WSig = <0.001) testing. Conclusions Cloacal swabs do not approximate either the or diversity of cecal samples, based on a paired sample analysis. The high variability of cloacal microbiota has been reported previously, and this study provides additional evidence of the randomness of cloacal microbiota in contrast to cecal microbiota. Our findings indicate that cloacal samples are not suitable for longitudinal studies of gut microbiota patterns. High inter-individual variation of cloacal swab data warrants further assessment of their reliability as a targeted diagnostic method.