Climate changes over the past 25 years have led to conducive conditions for invasive and transboundary fungal disease occurrence, including the re-emergence of wheat stem rust disease, caused by Puccinia graminis f.sp. tritici (Pgt) in East Africa, Europe, and the Mediterranean basin. Since 2018, sporadic infections have been observed in Tunisia. In this study, we investigated Pgt occurrence at major Tunisian wheat growing areas. Pgt monitoring, assessment, and sampling from planted trap nurseries at five different locations over two years (2021 and 2022) revealed the predominance of three races, namely TTRTF (Clade III-B), TKKTF (Clade IV-F), and TKTTF (Clade IV-B). Clade III-B was the most prevalent in 2021 as it was detected at all locations, while in 2022 Pgt was only reported at Beja and Jendouba, with the prevalence of Clade IV-B. The low levels of disease incidence during these two years and Pgt population diversity suggest that this fungus most likely originated from exotic incursions and that climate factors could have caused disease establishment in Tunisia. Further evaluation under the artificial disease pressure of Tunisian wheat varieties and weather-based modeling for early disease detection in the Mediterranean area could be helpful in monitoring and predicting wheat stem rust emergence and epidemics.