Introduction: Salmonella pathogenicity islands (SPIs) are regions scattered along the bacterial chromosome, with an acknowledged pivotal role during gastrointestinal and systemic infection. The distribution of SPIs has been investigated in reference strains. However, there is a lack of studies on their presence and/or assortment within the genomes of Salmonella enterica (S. enterica) serovars that circulate in different geographical regions. Therefore, in this study, we aimed to determine the presence of genes of the pathogenicity islands 1 to 5 (SPI-1 to 5), in Salmonella clinical isolates from Colombian patients with systemic and enteric outcomes. Methodology: A total of 125 strains of S. enterica belonging to different serovars were isolated from various clinical samples. Strains were identified and screened for the presence of various genes located in pathogenicity islands. The genes tested were selected according to the attributed pathogenic function and detected by PCR for the SPI-1 hilA and invA; for SPI-2 spiC and ttrC; for SPI-3 misL and mgtC; for SPI-4 orfL and SPI-4R; and for SPI-5 pipD and sopB. Results: Salmonella pathogenicity islands 1 to 5 were detected in isolates from patients with systemic and gastrointestinal infection. All the systemic isolates possessed all the genes tested; in contrast, 16 isolates from stool samples lacked one or more sequences encoded by the SPI-3 and SPI-4 (p < 0.000001). Conclusions: These results describe the heterogeneous distribution of SPIs-encoded sequences within the genomes of Colombian clinical isolates, and reveal important differences among systemic and stool sample isolates.