Bovine salmonellosis is an important contagious disease that is usually endemic to a particular region. the present study aimed to study the clinical cases, diagnostic tools and the specific gene responsible for the pathogenesis in Basrah, Iraq. The study was conducted on 2-4 years-old (88) native cow breeds from November 2022 to July 2023. Nearly, 38.63% of cows were positive for Salmonella by clinical signs, culture and confirmed using polymerase chain reaction (PCR). Diseased cows suffered from diarrhea, dehydration and pyrexia. Hematological analysis indicated leukocytosis, neutrophilia and significantly higher erythrocyte sedimentation rate and clotting factor indices. Results also indicated hypoglycemia, decreased total protein, and increased blood urea nitrogen. Fecal samples (1gm) were taken directly from the rectum of all suspected animals and transmitted immediately to the diagnostic lab in a cooling box. The PCR amplification and sequencing results of the 16S rRNA gene from Salmonella isolates revealed that 10 out of 19 isolates exhibited distinct bands of approximately 1500 bp corresponding to the Salmonella enterica serovar typhimurium. The results of cdtb and pltB genes amplification exhibited clear bands of approximately 819 bp and 518 bp, respectively. According to the phylogenetic analysis, our local isolates represented a close relationship to the other published strains in the NCBI database. In conclusion, bovine salmonellosis is considered a serious disease due to its negative effects on the infected animals of all ages. Therefore, early elimination and control of this disease could be the right way to protect farm animals.