Salmonella is an important animal and human pathogen responsible for Salmonellosis, and it is frequently associated with the consumption of contaminated poultry products. The aim of this study was to estimate the prevalence of Salmonella in the poultry farms and to determine the genetic relationship. A total of 135 samples collected from fifteen broiler farms, including cloacal, feed, water, environmental and farm operator faeces samples were subjected to microbiological isolation. Molecular confirmation of Salmonella isolates was carried out by amplification of the invA gene, discrimination of d-tartrate-fermenting Salmonella isolates using multiplex PCR, and subsequently analysed by pulsed-field gel electrophoresis (PFGE). A survey questionnaire was conducted to identify potential risk factors for Salmonella presence in broiler farms. The prevalence of Salmonella at the farm level was 26.67%, and Salmonella isolates were serotyped as S. Paratyphi B and all isolates were d-tartrate-fermenting (dT+). PFGE showed three highly similar clusters and one significantly different Salmonella isolate. S. Paratyphi B continued to be present in different links of the poultry chain in the Tolima region, and identification of its main source is necessary to control its dissemination.