In addition to their location at terminal positions, telomeric-like repeats are also present at internal sites of the chromosomes (intrachromosomal or interstitial telomeric sequences, ITSs). According to their sequence organization and genomic location, two different kinds of ITSs can be identified: (1) heterochromatic ITSs (het-ITSs), large (up to hundreds of kb) stretches of telomeric-like DNA localized mainly at centromeres, and (2) short ITSs (s-ITSs), short stretches of telomeric hexamers distributed at internal sites of the chromosomes. Het-ITSs have been only described in some vertebrate species and they probably represent the remnants of evolutionary chromosomal rearrangements. On the contrary, s-ITSs are probably present in all mammalian genomes although they have been described in detail only in some completely sequenced genomes. Sequence database analysis revealed the presence of 83, 79, 244 and 250 such s-ITSs in the human, chimpanzee, mouse and rat genomes, respectively. Analysis of the flanking sequences suggested that s-ITSs were inserted during the repair of DNA double-strand breaks that occurred in the course of evolution. An extensive comparative analysis of the s-ITS loci and their orthologous ‘empty’ loci confirmed this hypothesis and suggested that the repair event involved the direct action of telomerase. Whereas het-ITSs seem to be intrinsically prone to breakage, the instability of s-ITSs is more controversial. This observation is consistent with the hypothesis that s-ITSs are probably not themselves prone to breakage but represent ‘scars’ of ancient breakage that may have occurred within fragile regions. This study will review the current knowledge on these two types of ITS, their molecular organization, how they arose during evolution, their implications for chromosomal instability and their potential applications as phylogenetic/forensic markers.