Accessibility and transportation possibilities are key factors influencing societal conditions and land use patterns in rural areas. Thus, information on the spatial patterns of accessibility and transportation can be of paramount importance in understanding regional differences in development, human livelihood and land use patterns. Analysing spatio-temporal transportation patterns is particularly challenging in areas where everyday transportation is based on unscheduled public transportation and a naturally controlled seasonal transportation network, such as rivers. Here, our aim is to provide information on the seasonal dynamics of riverine transportation and its effects on accessibility patterns in Peruvian Amazonia. We analysed riverine transportation speeds using a low-cost GPS-based riverboat observation system. Spatio-temporal accessibility patterns were generalised from the GPS-observations that were classified according to seasons into the high water season, intermediate season and low water season. We show that navigation along the rivers has a clear seasonal and directional (upstream/downstream) variation, which varies with different types of rivers based on channel morphology. In addition, we conducted interviews with local people to study their perceptions of the seasonal changes in navigation and the accuracy of transportation schedules. As the travel distances in Peruvian Amazonia are generally long, seasonal and directional differences have clear impacts on the overall accessibility patterns in the area and on the livelihoods of riverine inhabitants. Furthermore, the lack of clearly scheduled transportation causes considerable uncertainty about transportation options for local communities. The baseline information of the seasonal and directional variation of riverine transportation and travel speeds provided by our work is usable in further accessibility and livelihood analyses for Peruvian Amazonia, but it may also be useful in other areas relying on riverine transportation.