Studies on the genetic diversity of coffee trees are important, considering their role in the maximization of productivity and quality. However, the success of a breeding program depends on the existence of genetic variability in the population under study. Our study aimed to evaluate the genetic diversity of the morpho-agronomic and anatomical leaf traits of different cultivars of Coffea arabica grown in the Gorongosa mountain region, in the area of Gorongosa National Park, Mozambique. The experiment assessed nine coffee cultivars based on their morpho-agronomic and anatomical traits. The plagiotropic branch diameter, leaf dry mass, leaf mass per area, number of epidermal cells + stomata, and stomatal form indicated differences among the studied cultivars. Among the nine C. arabica cultivars grown in the mountainous region of Gorongosa National Park, Mozambique, low genetic variability in morpho-agronomic traits was detected, while the genetic variability in leaf anatomical traits was higher. The nine cultivars were segregated into two groups, one consisting of Catucaí Amarelo 2SL and Catuaí Vermelho IAC 81, and the second consisting of Catuaí Amarelo IAC 39, Catucaí Vermelho 19/8, Acauã, Catucaí 785-15, Costa Rica, Catimor 128, and Catuaí Vermelho IAC 44. The cultivar segregation into the two groups indicated that the morpho-anatomical traits can be considered during the selection stages in breeding programs.