The fragmentation of DNA in historical specimens is very common, so obtaining sequences that allow molecular identification and the study of diversity is quite challenging. In this study, we used preserved and fresh specimens of the fruit fly genus Anastrepha, a genus of economic impact of fruit crops of the Neotropic. From these specimens, we evaluated: (1) the success PCR amplification rates of mini-barcodes fragments of the cytochrome c oxidase subunit I (COI) gene, and (2) the usefulness of mini-barcodes in the reconstruction of haplotypes for the identification of species and the diversity analysis. We used 93 specimens from 12 species, which had been preserved in 70% ethanol for more than 20 years. Internal primers were designed in the COI region and primers available in the literature were also evaluated. We obtained amplifications for 62.36% of the samples processed, and reconstructed haplotypes between 171 bp and 632 bp. Variable amplification rates between combinations of primers and between species were obtained, and molecular identification of some museum specimens was achieved. It was also possible to compare the haplotypes obtained in four species from which both fresh and museum samples were available. Our results also show the importance of the adjustment of the primers for the amplification, allowing to amplify fragments of up to 400 bp. The use available resources in biological collections is key to increasing knowledge of species of interest, and by means of the amplification of mini-barcodes, short sequences can be obtained that allow the molecular identification of specimens and the reconstruction of haplotypes with multiple purposes.