Background/Objectives: The classification and phylogenetic relationships of Gossypium hirsutum L. landraces, despite their proximity to southern Mexico, remain unresolved. This study aimed to clarify these relationships using SSR markers and hybridization methods, focusing on subspecies and race differentiation within G. hirsutum L. Methods: Seventy polymorphic SSR markers (out of 177 tested) were used to analyze 141 alleles and calculate genetic distances among accessions. Phylogenetic relationships were determined using MEGA software (version 11.0.13) and visualized in a phylogenetic tree. ANOVA in NCSS 12 was used for statistical analysis. Over 1000 inter-race crosses were conducted to assess boll-setting rates. Results: Distinct phylogenetic patterns were identified between G. hirsutum subspecies and races, correlating with boll-setting rates. Latifolium, richmondii, and morilli showed no significant increase in boll-setting rates in reciprocal crosses. Cultivars Omad and Bakht, as paternal parents, yielded higher boll-setting rates. Religiosum and yucatanense displayed high boll- and seed-setting rates as maternal parents but low rates as paternal parents. Additionally, phylogenetic analysis revealed a close relationship between cultivars ‘Omad’ and ‘Bakht’ with G. hirsutum race richmondii, indicating their close evolutionary relationship. Conclusions: Reciprocal differentiation characteristics of G. hirsutum subspecies and races, particularly religiosum and yucatanense, should be considered during hybridization for genetic and breeding studies. Understanding the phylogenetic relationships among G. hirsutum taxa is crucial for exploring the genetic diversity of this economically important species.