In Colombia, high genetic variability has been found among Trypanosoma cruzi stocks isolated from different vector and host species, using isoenzyme analysis and RFLP of total kinetoplastid DNA (kDNA), suggesting that several genetically related T. cruzi populations might be present within a single geographical area or adjacent ones. The objective of this study was to use the low-stringency single primer (LSSP)-PCR technique on variable regions of kDNA minicircles of T. cruzi to determine possible genetic relationships among stocks from distinct geographical regions of Colombia and different vector species and hosts. Although LSSP-PCR analysis showed a high genetic variability among 30 Colombian T. cruzi stocks, 29 of them belonged to T. cruzi lineage I, confirming that this lineage is predominant in different vector and host species from Colombia. Interestingly, one stock isolated from a Pastrongylus geniculatus bug was identified as T. cruzi lineage IIb, using PCR strategies targeted to the intergenic region of miniexon genes, a sequence encoding the D7 domain of the 24salpha ribosomal genes and the A10 fragment, being this finding, the first description of this lineage in Colombia. The LSSP-PCR signatures allowed correlation of most isolates with their respective geographical origins, and in one case from host and vector specimens at a same region, suggesting a transmission event. Moreover, variations in LSSP-PCR profiles among T. cruzi I stocks from a same region suggest that they may have a multiclonal character. Our results show that LSSP-PCR is a fast, valuable technique for characterization of intra-lineage polymorphism among T. cruzi stocks.