Cucurbita maxima Duch. is one of the most morphologically variable cultivated species. The Center for Conservation and Breeding of the Agricultural Diversity (COMAV) holds a diverse germplasm collection of the Cucurbita genus, with more than 300 landraces of this species. Morphological and molecular characterization are needed to facilitate farmer and breeder use of this collection. With this aim, the morphological variation of a collection of 120 C. maxima accessions was evaluated. The majority of these accessions originated from Spain, which has acted as a bridge since the 16th century for spreading squash morphotypes between the Americas and Europe. South American landraces (the center of origin of this species) were also included. Eight morphological types were established based on this characterization and previous intraspecific classifications. A subset of these accessions, selected from these classification and passport data, was employed for molecular characterization. Two marker types were used; sequence related amplified polymorphism (SRAP), which preferentially amplifies open reading frames (ORF), and amplified fragment length polymorphism (AFLP). In the main, SRAP marker analysis grouped accessions in accordance to their type of use (agronomic traits) and AFLP marker analysis grouped accessions as to their geographical origin. AFLP marker analysis detected a greater genetic variability among American than among Spanish accessions. This is likely due to a genetic bottleneck that may have occurred during the introduction of squash into Europe. The disparity of the results obtained with the two markers may be related to the different genome coverage which is characteristic of each particular marker type and/or to its efficiency in sampling variation in a population.