Background: In the field of spatial cognition, the study of individual differences represents a typical research topic. Gender and age have been prominently investigated. A promising statistical technique used to identify the different responses to items in relation to different group memberships is the Differential Item Functioning Analysis (DIF). The aim of the present study was to investigate the DIF of the Landmark positioning on a Map (LPM) task, across age groups (young and elderly) and gender, in a sample of 400 healthy human participants. Methods: LPM is a hometown map completion test based on well-known and familiar landmarks used to assess allocentric mental representations. DIF was assessed on LPM items two times: on categorical (i.e., positions) and coordinate (i.e., distances) scores, separately. Results: When positions and distances were difficult to assess with respect to the intended reference point, the probability to endorse the items seemed to get worse for the elderly compared to the younger participants. Instead other features of landmarks (high pleasantness, restorativeness) seemed to improve the elderly performance. A gender-related improvement of probability to endorse distance estimation of some landmarks, favoring women, emerged, probably associated with their repeated experiences with those landmarks. Overall, the complexity of the task seemed to have a differential impact on young and elderly people while gender-oriented activities and places seemed to have a differential impact on men and women. Conclusions: For the first time DIF was applied to a spatial mental representation task, based on the schematic sketch maps of the participants. The application of DIF to the study of individual differences in spatial cognition should become a systematic routine to early detect differential items, improving knowledge, as well as experimental control, on individual differences.