Abstract Objectives The Y chromosome has highly informative markers, such as single‐nucleotide polymorphisms (SNPs), that are useful for making historical inferences about the settlement of the Americas. However, the scarcity of these markers has limited their use. This study aims to identify new SNPs and increase the phylogenetic resolution of haplogroup Q for the Americas, mainly focusing on the lineages of the Amazon region. Materials and Methods Next‐generation sequencing was performed on two Y chromosomes belonging to haplogroup Q‐M3 using samples with divergent short tandem repeat haplotypes from the Colombian Amazon, and 14 of the new variants identified were selected for characterization in 207 samples of indigenous Colombians belonging to haplogroup Q‐M3. Results This methodology allowed us to establish nine new lineages within Q‐M3, including its paragroups. The most basal lineages were predominant in communities of Andean origin, such as the Embera‐Katio, the Nasas, and the Pastos. In contrast, the most distal lineages were restricted to inhabitants of the Amazon region of Vaupés. Discussion The SNPs reported here advance the development of subhaplogroups of Q‐M3 with a higher level of phylogenetic resolution than has been previously reported, which allowed the differentiation between populations that inhabit two regions of Vaupes area: the Pirá‐Paraná region and the upper and middle sections of the Vaupés River, and the region encompassing the Papurí River and the lower Vaupés. They are very useful for the microevolutionary analysis of the Amerindian populations of Colombia and of the Americas.