Morphological characterization of Animal Genetic Resources is the first step to documenting diversity and designing breed specific breeding programs. The current study characterized the morpho-biometric variation of indigenous chicken ecotypes prevailing in northwestern Ethiopia. A multi-stage purposive, stratified, and random sampling method was employed to select the study areas and chickens. A total of 1200 adult chickens were sampled and characterized for 12 qualitative and 11 quantitative traits. Univariate and multivariate data analysis methods were employed to analyze the data using SAS and R statistical software. Red plumage colour (33.2%), white and red earlobe colour (73.8%) and yellow shank colour (57.0%) were the most predominant colour trait categories. Sex, agro-ecology, location, and the interaction of sex and location had a highly significant (p<0.001) effect on all body measurements. Shank traits were found to have the highest discriminating power in both sexes. The overall classification rates for the female and male sample populations were 57.47% and 69.97%, respectively. The squared Mahalanobis distances between sites were significant (p<0.001) for both sexes. The longest distance was obtained between North Achefer and Banja (19.25) and between North Achefer and Dembecha (16.80) in female and male chickens, respectively. In female chickens, canonical variates 1 (CAN 1) and 2 (CAN 2) explained 82% of total variation and distinctly separated the sample populations of North Achefer and Jawi from others. In male chickens, 90% of the total variance is explained by CAN1, CAN2, and CAN3, which distinctly separate the sample populations of the North Achefer, Sinan, and Jawi, among others. Using cluster analysis, the indigenous chickens found in the study area could be classified into four ecotypes: ecotype 1 (Banja, Dembecha, and Aneded), ecotype 2 (North Achefer), ecotype 3 (Sinan), and ecotype 4 (Jawi).