Most studies of the genus Opuntia have focused on economically important species, and therefore more knowledge concerning the genetic diversity among wild and locally managed Opuntia species is needed for an expanded use of cacti in the future. The present study is part of ongoing ethnobotanical work in the Tehuacán-Cuicatlán Valley of Mexico and focuses on six traditionally classified forms of Opuntia pilifera used as food by the indigenous Popoloca people in San Juan Atzingo. Traditional knowledge of how to distinguish these forms based on fruit flavor, color, size, and number of spines on the fruits and cladodes is preserved in the local community. Genetic fingerprinting with 129 AFLPs did not correlate with this traditional morphological classification of 67 cacti. Yet, these AFLPs distinguished the analyzed 67 Opuntia pilifera cacti easily from the out-group comprising 17 wild Opuntia velutina.