Morphological characters (six traits) and isozymes (four systems, five loci) were used to discriminate between Spanish chestnut cultivars (Castanea sativa Mill.) from the Iberian Peninsula. A total of 701 accessions (representing 168 local cultivars) were analyzed from collections made between 1989 and 2003 in the main chestnut growing areas: 31 were from Andalucía (12 cultivars), 293 from Asturias (65 cultivars), 25 from Castilla-León (nine cultivars), four from Extremadura (two cultivars) and 348 from Galicia (80 cultivars). Data were synthesized using multivariate analysis, principal component analysis, and cluster analysis. A total of 152 Spanish cultivars were verified: 58 cultivars of major importance and 94 of minor importance, of which 18 had high intracultivar variation. Thirty-seven cultivars were clustered into 14 synonymous groups. Six of these were from Galicia, one from Castilla-León (El Bierzo), four from Asturias, one from Asturias and Castilla-León (El Bierzo), and two from Asturias, Castilla-León (El Bierzo), and Galicia. The chestnut cultivars from Galicia and Asturias were undifferentiated in genetic terms, indicating that they are not genetically isolated. Overall, chestnut cultivars from southern Spain showed the least variation. Many (58%) of Spanish cultivars produced more than 100 nuts/kg; removing this low market-value character will be a high priority. The data obtained will be of use in chestnut breeding programs in Spain and elsewhere.