Molecular cytogenetic reveal details of fine chromosomal structure that are useful in studies of chromosomal diversification, genome organization, and evolution. However, these techniques have rarely been used to describe population-level variations in the tribe Meliponini. In the present study, we compared the karyotype, number, and localization of rDNA clusters and the distribution of a repetitive DNA sequence in the genome of T. spinipes from nine locations. Although karyotypes with 2n = 34 were found in all cases, five different karyotypic formulae were detected. The heterochromatin was preferentially associated with centromeric regions. The microsatellite probe (GA)15 signals were localized mainly in the euchromatic regions of all chromosomes in most of the analyzed populations. The 18S rDNA clusters were present in four chromosomal pairs, except for the population from Palotina, which showed only two rDNA clusters. Analyses revealed a karyotypic variation in T. spinipes along the geographic distribution, proving that cytogenetic variations were not detected when scattered colonies were analyzed.