We present a mitochondrial DNA (mtDNA) haplotype phylogeny for Amazonian Anolis lizards, including geographical sampling within four species distributed across the Amazon basin (A. fuscoauratus, A. nitens, A. ortonii and A. punctatus). Approximately 1500 bp of mtDNA encoding ND2, COI and four transfer RNAs (tRNAs) are reported for 39 specimens representing four to five populations of each widespread species, plus eight outgroups. These new sequences are aligned with eight previously published sequences, yielding 914 variable characters and 780 parsimony-informative characters. Phylogenetic analyses using maximum parsimony and maximum likelihood reject the hypothesis that Amazonian anoles form a monophyletic group excluding Central American and Caribbean anoles, and suggest multiple faunal exchanges among these regions. Haplotype divergence among geographical populations of A. nitens, whose variation was influential in formulating the Pleistocene refuge hypothesis of Amazonian speciation, is very large (13–22% sequence difference), suggesting that these populations separated well before the Pleistocene. Haplotype divergences among geographical populations of A. fuscoauratus (3–4%), A. punctatus (4–9%) and A. ortonii (6–8%) also indicate pre-Pleistocene differentiation within each species, but temporally incongruent patterns among species.