This study aims to portray the complex diversity of the Mexican Mestizo population, which represents 98.8% of the entire population of Mexico. We compiled extended haplotype data of the Y chromosome from populations in the Central Valley of Mexico (CVM), which we compared with other Mestizo and parental (Amerindian, European, and African) populations. A complex ancestral relationship was found in the CVM population, suggesting cosmopolitan origins. Nevertheless, the most preeminent lineages point toward a European ancestry, where the R1b lineage was most frequent. In addition, important frequencies of Amerindian lineages were also found in the Mestizo sample studied. Interestingly, the Amerindian ancestry showed a remarkable substructure, which was represented by the two main founding lineages: QL54 (× M3) and M3. However, even within each lineage a high diversity was found despite the small number of sample bearers of these lineages. Further, we detected important genetic differences between the CVM populations and the Mexican Mestizo populations from the north and south. This result points to the fact that Mestizo populations present different ancestral proportions, which are related to the demographic events that gave origin to each population. Finally, we provide additional forensic statistical parameters that are useful in the interpretation of genetic analysis where autosomal loci are limited. Our findings illustrate the complex genetic background of the Mexican Mestizo population and reinforce the need to encompass more geographic regions to generate more robust data for forensic applications.