Background: Gut dysbiosis is strongly associated with obesity and T2DM pathogenesis. Objective: To study the possible associations of gut microbiota 16s RNA target region with obesity. Patients and Methods: A case-control study that was done on 60 subjects (20 obese subjects without diabetes, 20 obese diabetic patients, and 20 control normal subjects). This study was based on gut microbiota 16s rRNA detection by DNA extraction from fresh frozen stool samples of study groups. Gene amplification was done by PCR. Using gene sequencing to identify the gut microbiota species. Results: 2 cases were positive for 16s RNA gene in control group, the sequence of this gene was belonging to Succinivibrio dextrinosolvens. 3 cases were positive for 16s RNA gene in obese without diabetes group; all were belonging to Prevotella copri. 5 cases were positive for 16s RNA gene in obese with diabetes group; 3 cases were belonging to Prevotella copri and 2 cases were belonging to Succinivibrio dextrinosolvens. Statistically there was no significant difference among the three study groups regarding the 16s RNA gene, nor abundant organism. Although there was a significant difference between obese without DM group & lean group regarding type of organism as 100% of obese without DM group had Prevotella copri compared to 0% in group A (P = 0.04). However, there was no statistical difference between lean group and obese with DM group as (p = 0.20). In addition, there was no difference between obese group and obese with DM group (P= 0.27) regarding type of organisms. Conclusion:Obesity is associated with microbial composition alteration using 16s rRNA sequences method.