Aim Our aim was to investigate genetic structure in Neotropical populations of common green iguanas (Iguana iguana) and to compare that structure with past geological events and present barriers. Additionally, we compared levels of divergence between lineages within Iguana with those within closely related genera in the subfamily Iguaninae. Location Neotropics. Methods DNA sequence data were collected at four loci for up to 81 individuals from 35 localities in 21 countries. The four loci, one mitochondrial (ND4) and three nuclear (PAC, NT3, c‐mos), were chosen for their differences in coalescent and mutation rates. Each locus was analysed separately to generate gene trees, and in combination in a species‐level analysis. Results The pairwise divergence between Iguana delicatissima and I. iguana was much greater than that between sister species of Conolophus and Cyclura and non‐sister species of Sauromalus, at both mitochondrial (mean 10.5% vs. 1.5–4%, respectively) and nuclear loci (mean 1% vs. 0–0.18%, respectively). Furthermore, divergences within I. iguana were equal to or greater than those for interspecific comparisons within the outgroup genera. Phylogenetic analyses yielded four strongly supported, geographically defined mitochondrial clades (3.8–5% divergence) within I. iguana. Three of the four clades were found using PAC (0.18–1.65% divergence) and two using NT3 (0.6% divergence) alone. The primary divergence, recovered in three polymorphic loci, was between individuals north and south of the Isthmus of Panama. The southern group was differentiated into clades comprising individuals on either side of the northern Andes, using both PAC and ND4. Main conclusions Deep genetic divergences were found within I. iguana that are congruent with past and current geological barriers. These divisions are greater than sister species comparisons in other Iguaninae genera, indicating the possible presence of cryptic species. Geological changes from the mid‐Miocene through the Plio‐Pleistocene have shaped the pattern of divergence in I. iguana. The uplift of the northern Andes presented a barrier between South American I. iguana populations by 4 Ma. Populations north of the Isthmus of Panama form a clade that is distinct from those to the south, and may have expanded northwards following the closing of the Isthmus of Panama 2.5 Ma.