This study delves into the cytogenetic, molecular, and ecological aspects of the Alstroemeria pulchra complex, emphasizing the role of prior morpho-colorimetric analyses in differentiating the infraspecific taxa of A. pulchra. The integration of advanced morpho-colorimetric analyses provided a quantitative basis for this differentiation, showcasing significant variations in flower morphology and coloration that precede and complement the cytogenetic, molecular, and ecological findings presented herein. Cytogenetic studies unveiled chromosomal disparities, and differentiated genetic clusters support the taxonomic separation of A. pulchra var. maxima from A. pulchra var. pulchra and A. pulchra subsp. lavandulacea. Additionally, updated morphometric descriptions, observations, and a differentiation key for the subspecies of A. pulchra are provided. This integrative approach not only deepens the understanding of biodiversity and adaptation in Alstroemeria but also underscores the synergy between various scientific disciplines to unravel taxonomic complexities. The obtained results pose new questions about the evolution and adaptation in related Alstroemeria species, the methodologies utilized can serve as a significant contribution to future research and conservation of this botanical genus with ornamental and ecological value.