We investigate the enamel microstructure of 37 isolated rodent incisors from several late middle Eocene and late Oligocene localities of Contamana (Loreto Department, Peruvian Amazonia), and from the early Oligocene TAR-01 locality (Shapaja, San Martín Department, Peruvian Amazonia). All incisors show an enamel internal portion with multiserial Hunter-Schreger Bands (HSB). The late middle Eocene localities of Contamana yield incisors with subtypes 1, 1–2, and 2 of multiserial HSB; TAR-01 yielded incisors with subtypes 1–2, 2, 2–3, and 3 of multiserial HSB; and the late Oligocene localities of Contamana, incisors with subtypes 1–2, 2, and 2–3 of multiserial HSB. Based on our current knowledge of the South American and African rodent fossil records and given the primitiveness of the Eocene caviomorph faunas, it may be expected that the hystricognath pioneer(s) who have colonized South America from Africa sometime during the middle Eocene, most probably had incisors that displayed a multiserial enamel with an interprismatic matrix arrangement characterizing the subtype 1 (or subtype 1 + the subtype 2 and/or the transitional 1–2) of multiserial HSB. In contrast, the derived subtypes 2–3 and 3 conditions were subsequently achieved but likely rapidly, as evidenced by its record as early as the ?late Eocene/early Oligocene (e.g., Santa Rosa, Shapaja, and La Cantera), and seemingly evolved iteratively but only in the Octodontoidea clade. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.