Six distinct oil tribes were established using multivariate statistical analysis of source-related biomarker and isotopic ratios for 73 crude oil samples from the Middle Magdalena Valley (MMV), Colombia. These six tribes show a systematic distribution by both basin location and reservoir rock age and may originate from different source rocks or different organofacies of the same source rock. Biomarker and isotopic data further differentiate the tribes with respect to source rock depositional environment, lithology, organic matter type, and thermal maturity. The thermal maturity and reservoir interval for the northernmost tribe 5 suggest a middle Cretaceous Tablazo Formation source rock. In contrast, tribes 1 through 4 are likely derived from the primary regional source rock, the Upper Cretaceous La Luna Formation. However, we observe regional differences in bulk properties, thermal maturity, terrigenous input, and oxicity between the four La Luna–derived oil tribes. In addition, tribe 3 appears to result from end-member mixing between tribes 2 and 4. Finally, the southernmost tribe 6 is the only oil with terrigenous character. Diamondoid analysis shows the presence of significant secondary cracking in the tribe 6 oil with low levels of cracking present in oil samples from tribes 1 and 2 in the central MMV. This suggests a more deeply buried nonmarine source along the western flank of the Andean Eastern Cordillera. The integration of chemometric, biomarker, and diamondoid analyses have improved our understanding of the MMV petroleum system and advocate for the presence of three or more source rock intervals within the basin.