Single-grain optical dating has been widely used to estimate depositional ages for Quaternary sediments. An understanding of the sources influencing the dispersion of equivalent dose (De) estimates from individual grain measurements is essential for accurate age determination. Beta microdosimetry (i.e., beta dose deposition at the sub-millimetre scale) is a known cause of spread in single-grain De values, so the detection and assessment of beta dose rate (D˙β) variation is important to properly interpret these data. Here we demonstrate the application of Timepix, a pixelated detector that directly measures in situ beta emissions based on cluster analysis, to determine the beta microdosimetry of natural sediment samples from a cave deposit in Russia and a sand dune in Australia. We describe a Timepix measurement and data processing procedure for natural sediments, and establish sample-specific calibration curves using associated sample radioelement concentrations to convert Timepix count rates into estimates of D˙β. On the basis of the Timepix analysis, a 2D ‘heat map’ of D˙β at sub-millimetre resolution was obtained for each sample. Our results show that the D˙β estimates are heterogeneous and their non-uniformity gives rise to 9 ± 4 to 26 ± 5% overdispersion in the single-grain De distributions for the samples examined here. We discuss the likely sources of D˙β heterogeneity, based on micromorphological investigations of these sediment samples, which include a variety of materials present in natural deposits associated with D˙β ‘hot’ and ‘cold’ spots. A comparison of the D˙β dispersion with that of the corresponding single-grain De values shows that the scatter among the latter can be fully or partly explained by beta microdosimetry and other known sources of overdispersion.