The discovery of the role of light exposure for the development of lateralization in domestic chick embryos revolutionized this research field. However, two main issues remain unresolved: (i) while in chicks anatomical light-dependent lateralization is mostly confined to the thalamofugal visual pathway, in pigeons only the tectofugal pathway is lateralized after light exposure. However, no study in either species ever investigated anatomical lateralization in the entopallium, the forebrain station of the tectofugal pathway. (ii) It is now known that lateralization can be observed also in dark-incubated chicks, both at the behavioural and at the Immediate Early Gene-expression level. We hypothesized that lateralization of the tectofugal system may underlie these light-independent effects. To investigate structural lateralization in the tectofugal pathway of dark-incubated chicks, we used parvalbumin (PV) as a marker of a sub population of entopallial neurons, quantifying PV-ir cell densities in the left and right entopallium. We found higher density in the right hemisphere, revealing for the first time anatomical lateralization in entopallium and confirming its potential role in supporting lateralized brain processing in dark-incubated birds. Results are discussed in relation to the possible functional role of PV-ir cells in inhibitory neural functions.