This study was embarked upon to characterise phenotypically helmeted guinea fowls in three agro-ecologies in Nigeria using multivariate approach. Eighteen biometric characters, four morphological indices and eleven qualitative (phaneroptic) traits were investigated in a total of 569 adult birds (158 males and 411 females). Descriptive statistics, non-parametric Kruskal–Wallis H test followed by the Mann–Whitney U test for post hoc, Multiple Correspondence Analysis (MCA), General Linear Model, Canonical Discriminant Analysis, Categorical Principal Component Analysis and Decision Trees were employed to discern the effects of agro-ecological zone and sex on the morphostructural parameters. Agro-ecology had significant effect (P<0.05; P <0.01) on all the colour traits. In general, the most frequently observed colour phenotype of guinea fowl had pearl plumage colour (54.0%), pale red skin colour (94.2%), black shank colour (68.7%), brown eye colour (49.7%), white earlobe colour (54.8%) and brown helmet colour (72.6%). The frequencies of helmet shape and wattle size were significantly influenced (P <0.01) by agro-ecology and sex. Overall, birds from the Southern Guinea Savanna zone had significantly higher values (P <0.05) for most biometric traits compared to their Sudano-Sahelian and Tropical Rainforest counterparts. They were also more compact (120.83±1.61 vs. 113.96±0.97 vs. 111.33±1.19) and had lesser condition index (8.542±0.17 vs. 9.92±0.10 vs. 9.61±0.13) than their counterparts in the two other zones. The interaction between agro-ecology and sex had significant effect (P <0.05) on some quantitative variables. The MCA and discriminant analysis revealed considerable intermingling of the phaneroptic, biometric traits and body indices especially between the Sudano-Sahelian and Tropical Rainforest birds. Inspite of the high level of genetic admixture, the guinea fowl populations could best be distinguished using wing length, body length and eye colour. However, further complementary work on genomics will guide future selection and breeding programmes geared towards improving the productivity, survival and environmental adaptation of indigenous helmeted guinea fowls in the tropics.