Bess beetle species tend to be endemic and are useful indicators for the designation of conservation areas. However, little is known about the diversity and distribution of these beetles in El Salvador. Here, we present results from a study testing if species segregated along the altitudinal gradient of Montecristo National Park, El Salvador. The survey was done between September and December 2015. Beetles were collected at nine transects from three ecosystems, which included cloud, mixed-pine, and dry forests that occur along the altitudinal gradient of Montecristo. We sampled a total of 696 trees, finding 79 galleries of bess beetles, belonging to 13 species, which included: Arrox agassizi (Kaup), Chondrocephalus granulifrons (Bates), Chondrocephalus sp., Chondrocephalus salvadorae (Schuster), Heliscus eclipticus (Truqui), Odontotaenius striatopunctatus (Percheron), Ogyges politus (Hincks), Oileus sargi (Kaup), Passalus punctatostriatus Percheron, Passalus punctiger Lepeletier & Audinet-Serville, Verres hageni Kaup, Vindex sculptilis Bates, and Chondrocephalus gemmae Reyes-Castillo & Castillo which is a new species record for El Salvador. We found that species segregated by altitude, with the highest species richness observed in the cloud forest (n = 6), followed by the dry (n = 5) and mixed pine-oak (n = 4) forests. Most species were randomly associated with different tree species trunks, with the exception of V. hageni which was associated with trunks of Cecropia sp. Loefl. (Rosales: Urticaceae), and P. punctastriatus and P. punctiger which were associated with trunks of Cordia alliodora (Ruiz & Pav.) Oken (Boraginales: Boraginaceae). Our results show that species segregate by habitat, as only two species (C. gemmae and H. eclipticus) were found in more than one ecosystem.