In the highland regions of Ethiopia the heterogeneity of the land, the climate, and soil favors the presence of a large number of landraces. We analyzed a representative sample of 62 traditional Ethiopian highland maize accessions, using amplified fragment length polymorphism (AFLP®) markers and morphological traits with the aim to group the accessions based on the their genetic profiles and morphological traits, to study agroecological variation and to assess the level of correlation between phenotypic and genetic distances. Eight EcoRI/MseI primer combinations and 15 morphological traits were used. The accessions varied significantly for all of the measured morphological traits. Of a total of 650 AFLP markers that were scored, 89.5% were polymorphic. Pair-wise genetic distance estimates based on AFLP data revealed dissimilarity coefficients ranging from 0.32 to 0.69 (mean of 0.57). Cluster analysis of the AFLP data grouped most accessions collected from the Northern highlands into one major cluster. It, however, failed to separate the Western and Southern accessions into different clusters. Regardless of the large variation in environmental conditions between agroecologies, only 9% of the total genetic variation was found between agroecologies, whereas 91% was found within agroecologies in Ethiopia. This finding may be explained by long distance seed exchange, continuous seed introduction and gene flow between agroecologies. The relationship between morphological and AFLP-based distances was significant and positive. Based on this study, three groups of highland accessions, with distinctive genetic profiles and morphological traits were identified. This information will be useful for further collections and conservation of the unique diversity included in the highland maize landraces of Ethiopia.