This study was conducted to characterise phenotypically helmeted Guinea fowls in three agro-ecologies in Nigeria using multivariate approach. Eighteen biometric characters, four morphological indices and eleven qualitative physical 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 and Dunn-Bonferroni tests for post hoc, Multiple Correspondence Analysis (MCA), Univariate Analysis, 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 (120.00 vs. 110.00 vs. 107.69) but had lesser condition index (7.66 vs. 9.45 vs. 9.30) and lower long-leggedness (19.71 vs. 19.23 vs. 9.51) than their counterparts from the two other zones. Sexual dimorphism (P<0.05) was in favour of male birds especially those in Southern Guinea Savanna and Sudano-Sahelian zones. However, the MCA and discriminant analysis revealed considerable intermingling of the qualitative physical traits, biometric traits and body indices especially between the Sudano-Sahelian and Tropical Rainforest birds. In spite of the high level of genetic admixture, the Guinea fowl populations could to a relative extent be distinguished using wing length, body length and eye colour. Generally, the birds from the three zones appeared to be more homogeneous than heterogeneous in nature. However, further complementary work on genomics will guide future selection and breeding programs geared towards improving the productivity, survival and environmental adaptation of indigenous helmeted Guinea fowls in the tropics.