We investigate the enamel microstructure of 37 isolated rodent incisors from several late middle Eocene and late Oligocenelocalities of Contamana (Loreto Department, Peruvian Amazonia), and from the early Oligocene TAR-01 locality (Shapaja, SanMartín 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-01yielded incisors with subtypes 1–2, 2, 2–3, and 3 of multiserial HSB; and the late Oligocene localities of Contamana, incisorswith subtypes 1–2, 2, and 2–3 of multiserial HSB. Based on our current knowledge of the South American and African rodentfossil 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 amultiserial enamel with an interprismatic matrix arrangement characterizing the subtype 1 (or subtype 1 + the subtype 2 and/orthe transitional 1–2) of multiserial HSB. In contrast, the derived subtypes 2–3 and 3 conditions were subsequently achieved butlikely 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.