Spanish speaking populations in the USA have long been categorised under the umbrella term ‘Hispanic’, which is a cultural construct. The term Hispanic ignores the unique ethnohistories and biological variation among Hispanic groups with various European, African and indigenous American influences. Considerable heterogeneity has been identified in pre-contact America and has continued to influence the cultural and biological compositions of various regions today. The purpose of this research is to examine biological variation in Mexico, which was influenced by indigenous migration patterns and the Old World conquests of the Americans. Using multivariate statistics, this paper compares 16 three-dimensional craniometric landmarks of samples from northern Central Mexico, northern Yucatan and western Mexico to examine the regional biological variation present in Mexico in both prehistoric and historic groups and also compares Mexican, Spanish and African American groups to examine patterns of Old World conquests. Multivariate statistics detected significant group differences for both size and shape (centroid size, p < 0.0001; shape, p < 0.0001) and showed that while significantly different, all the Mexican groups are more similar to one another except for one prehistoric inland-western Mexican group, which is morphologically distinct from the other Mexican groups. Previous mtDNA research in these areas shows a low prevalence of African American admixture and a high indigenous component in the northern Mexican groups, which is consistent with the findings of this paper. The prehistoric and historic Mexican groups were the most similar indicating the retention of indigenous admixture after contact. The results from this analysis demonstrate that all groups are significantly different from one another supporting other findings that have shown that the indigenous populations of the New World are heterogeneous and that this variation may also contribute to the heterogeneity of contemporary populations. Copyright © 2013 John Wiley & Sons, Ltd.