Forensic palynology is a discipline used in criminal cases, the importance of which has been increasing within the forensic sciences worldwide over the past three decades. Palynological analysis of surface soil samples collected from crime scenes, items and individuals has already been proven to provide important evidence linking suspects, victims and items to specific locations. A palynological study of surface soil obtained in two Portuguese districts, Coimbra and Setubal, was undertaken. The main aim was to determine the value of soil samples regarding the plant community diversity in a given area of the country, based on the evaluation of palynomorph assemblages, and to determine whether any variation could be useful in a forensic palynology context. Five surface soil samples were obtained and processed from three representative types of habitat (dunes, mixed forest and scrub) within the two districts, providing 30 samples. In total, 5434 palynomorphs were analysed and 62 taxa identified, representing nine families, 42 genera and 11 species. Results show that both districts were generally characterised by high pollen taxa frequencies, by composition diversity and by distinct palynological profiles for each district, area and collection site. In conclusion, this study shows that different locations varied in their pollen profiles, which may be of use to forensic palynologists.