The systematic study of the Plagiolophinae (Remy, 1976) nov. rank (Perissodactyla, Mammalia) from the Eocene of the Duero Basin (Castilla y Leon, Spain) has permitted the identification of two new species: Plagiolophus casasecaensis nov. sp. of the Casaseca site (middle Eocene, MP 13-14, Zamora province) that is a very primitive species; and Plagiolophus mazateronensis nov. sp. of the Mazateron (middle-upper Eocene, MP 16-17, Almazan subbasin, Soria province) and Caenes sites (middle Eocene, MP 16, Salamanca province) which are described by a special combination of primitive and derivative characteristics unknown in other species of the genus. Other forms poorly documented and attributable to genus Plagiolophus occur in Jambrina and El Viso-Sanzoles (MP 13-14, Zamora province), San Morales (MP 16, Salamanca province), Deza (upper Eocene, Soria province), Molino del Pico (upper Eocene-Oligocene, Zamora province), and Mazateron. The genus Leptolophus that has been determined in Mazateron, had only been known before in sorne French localities. This Plagiolophinae assemblage is clearly biogeographically different from the already known findings in occidental Europe and subpirenaic area.