Threespine stickleback were used to examine phenotypic plasticity of telencephala in relation to inferred ecology. Fish from derived, allopatric, freshwater populations were sampled from three shallow, structurally complex lakes with benthic-foraging stickleback (benthics) and from three deep, structurally simple lakes with planktivores (limnetics). The telencephalon of specimens preserved immediately after capture (field-preserved), field-caught fish held in aquaria for 90 days (lab-held), and lab-bred fish from crosses and raised in aquaria were compared. Field-preserved sea-run (ancestral) stickleback were collected from two separate sites, and parents of lab-bred sea-run stickleback were collected from one of these sites. In field-preserved and lab-held fish, the telencephala of limnetics exhibited triangular dorsal shape, while those of benthics and sea-run fish had rounder shapes. No such pattern was detected in lab-bred fish. Within each treatment type, benthics had larger relative telencephalon sizes, using overall brain size as the covariate, than limnetics. Among field-preserved samples, sea-run fish had smaller telencephalon sizes than lake fish. Intra-population analyses of lake samples showed that field-preserved fish consistently had larger relative telencephalon sizes than lab-bred fish. The opposite was true of the sea-run population. In a separate study using one benthic population and one limnetic population, samples were preserved in the field immediately or held in the lab for 30, 60, and 90 days before they were sacrificed. In both populations, the telencephalon shapes of lab-held fish were similar to those of field-preserved fish but became progressively more like lab-bred ones over 90 days. In contrast, relative telencephalon size decreased dramatically by 30 days after which there was little change. In freshwater threespine stickleback, the telencephalon exhibits considerable phenotypic plasticity, which was probably present in the ancestor [Current Zoology 58 (1): 189–210, 2012].