Cafeteria (CAF) diet is known to accurately mimic the human Western diet in modern societies, thereby inducing severe obesity accompanied by drastic alterations on the gut microbiome in animal models. Notably, the dietary impact in the gut microbiota composition might be influenced by genetic factors, thus distinctively predisposing the host to pathological states such as obesity. Therefore, we hypothesized that the influence of strain and sex on CAF-induced microbial dysbiosis leads to distinct obese-like metabolic and phenotypic profiles. To address our hypothesis, two distinct cohorts of male Wistar and Fischer 344 rats, as well as male and female Fischer 344 animals, were chronically fed with a standard (STD) or a CAF diet for 10 weeks. The serum fasting levels of glucose, triglycerides and total cholesterol, as well as the gut microbiota composition, were determined. CAF diet triggered hypertriglyceridemia and hypercholesterolemia in Fischer rats, while Wistar animals developed a marked obese phenotype and severe gut microbiome dysbiosis. Furthermore, CAF diet-induced changes on gut microbiota were related to more profound alterations in body composition of female than male rats. We revealed that distinct rat strains and genders chronically consuming a free-choice CAF diet develop distinct and robust microbiota perturbations. Overall, we showed that genetic background might have a key role in diet-induced obesity, thus distinguishing the suitability of different animal models for future nutritional studies focused on gut microbiota dysbiosis induced by a CAF dietary model.