We report Anopheles darlingi in Darien Province in eastern Panama. Polymerase chain reaction–restriction fragment length polymorphism profiles of the single copy nuclear white gene and sequence comparisons confirmed the presence of 66 specimens of the northern lineage of An. darlingi. The parsimony network depicted 5 CO1 haplotypes in 40 specimens of An. darlingi, which connected through 7–8 mutational steps with sequences from Central and South America. Furthermore, the presence of haplotypes in Biroquera, Darien Province identical to those previously published from northern Colombia suggests that Panamanian samples originated in Colombia. Results of neutrality tests (R2 and Fu’s FS) were not significant and the mismatch distribution was multimodal and did not fit the model of sudden population growth. These findings may indicate a long and stable presence of An. darlingi in eastern Panama.