Hydroelectric facilities often release water at variable rates over the day to match electricity demand, resulting in short-term variability in downstream discharge and water levels. This sub-daily variability, known as hydropeaking, has mostly been studied at large facilities. The ongoing global proliferation of small hydropower (SHP) facilities, which in Brazil are defined as having installed capacities between 5 and 30 MW, raises the question of how these facilities may alter downstream flow regimes by hydropeaking. This study examines the individual and cumulative effects of hydropower facilities on tributaries in the upland watershed of the Pantanal, a vast floodplain wetland system located on the upper Paraguay River, mostly in Brazil. Simultaneous hourly discharge measurements from publicly available reference and downstream gage stations were analyzed for 11 reaches containing 24 hydropower facilities. Most of the facilities are SHPs and half are run-of-river designs, often with diversion channels (headraces). Comparison of daily data over an annual period, summarized by indicators of hydrological alteration (HA) that describe the magnitude, frequency, rate of change, and duration of flows, revealed differences at sub-daily scales attributable to hydropeaking by the hydropower facilities. Results showed statistically significant sub-daily HA in all 11 reaches containing hydropower facilities in all months. Discharge indicators that showed the highest percentage of days with increased variability were the mean rates of rise and fall, amplitude, duration of high pulses, maximum discharge, and number of reversals. Those that showed higher percentages of decreased variability included minimum discharge, number of high pulses, duration of stability, and number of low pulses. There was no correlation between HA values and physical characteristics of rivers or hydropower facilities (including installed capacity), and reaches with multiple facilities did not differ in HA from those with single facilities. This study demonstrates that SHPs as well as larger hydropower facilities cause hydrological alterations attributable to hydropeaking. Considering the rapid expansion of SHPs in tropical river systems, there is an urgent need to understand whether the ecological impacts of hydropeaking documented in temperate biomes also apply to these systems.