Biological impacts of the Fukushima nuclear accident have been reported in various organisms but have not been evaluated sufficiently. Here, we collected the pale grass blue butterfly, Zizeeria maha, in September 2016, 5.5 years after the accident, from ex-evacuation zones in Fukushima Prefecture to evaluate the remaining biological effects of the accident. Although the morphological abnormality rate was not high, it was significantly positively correlated with the initial 131I and 137Cs deposition levels (as of March 2011). The capture rate was negatively correlated with the initial 137Cs deposition level at the borderline of significance. Neither the abnormality rate nor the capture rate was correlated with the ground radiation dose (as of September 2016). Generalized linear model (GLM) analysis also indicated contributions of the initial 131I and 137Cs deposition levels but not the ground radiation dose. These results suggest that the butterflies present in the ex-evacuation zones in 2016 were still affected, although not severely, mainly because of the initial high-dose exposure to radioiodine, radiocesium, and other radionuclides. We conclude that transgenerational effects of radioactive contamination have successively manifested in the 5.5 years since the accident, possibly through genetic mutations, although potential contributions of other modes of damage cannot be ignored.