Comparative cytogenetic studies on Xenarthra, one of the most basal mammalian clades in the Placentalia, are virtually absent, being restricted largely to descriptions of conventional karyotypes and diploid numbers. We present a molecular cytogenetic comparison of chromosomes from the two-toed (Choloepus didactylus, 2n = 65) and three-toed sloth species (Bradypus tridactylus, 2n = 52), an anteater (Tamandua tetradactyla, 2n = 54) which, together with some data on the six-banded armadillo (Euphractus sexcinctus, 2n = 58), collectively represent all the major xenarthran lineages. Our results, based on interspecific chromosome painting using flow-sorted two-toed sloth chromosomes as painting probes, show the sloth species to be karyotypically closely related but markedly different from the anteater. We also test the synteny disruptions and segmental associations identified within Pilosa (anteaters and sloths) against the chromosomes of the six-banded armadillo as outgroup taxon. We could thus polarize the 35 non-ambiguously identified chromosomal changes characterizing the evolution of the anteater and sloth genomes and map these to a published sequence-based phylogeny for the group. These data suggest a low rate of genomic repatterning when placed in the context of divergence estimates based on molecular and fossil data. Finally, our results provide a glimpse of a likely ancestral karyotype for the extant Xenarthra, a pivotal group for understanding eutherian genome evolution.