The aim of this study was to investigate whether Pleistocene climatic instability influenced the phylogeographic structure and historical demography of an endemic Atlantic Forest (AF) orchid bee, Euglossa iopoecila Dressler, which shows two main patterns of integument colors over of its geographical distribution. We based our analysis on the concatenated sequence of four mtDNA segments belonging to genes 16S (357 bp), Cytb (651 bp) and COI (1206 bp), totaling 2234 bp. Samples of E. iopoecila populations were collected in 14 AF remnants along its geographic distribution. Median-Joining haplotype networks, SAMOVA and BAPS results indicated three lineages (southern, central and northern clusters) for E. iopoecila, with two important phylogeographic ruptures. We found higher genetic diversity among samples collected in the central region of the AF, which coincides with predicted areas of climatic stability, according to recent AF stability–extinction model. The demographic analysis suggests that only the southern cluster had undergone recent population expansion, which probably started after the last glacial maximum (LGM). Our data suggest that the differentiation observed in the three mitochondrial lineages of E. iopoecila is the result of past disconnections and multiple extinction/recolonization events involving climate fluctuations. In terms of conservation, we would emphasize the importance of considering: (1) the region of the central clade as the location of the highest genetic diversity of mtDNA of E. iopoecila populations; (2) the philopatric behavior of females that tends to restrict mtDNA gene flow in particular, with direct implications for the conservation of the total genetic diversity in euglossine populations. © 2016 Springer Science+Business Media Dordrecht183539552