The Karoo region of South Africa is an ideal laboratory to use ambient seismic signals to map the shallow subsurface, as it is a quiet and pristine environment with a geology that is relatively well known. Ambient seismic signals were continuously recorded for a ten week period between August and October 2015. The ambient seismic noise network consisted of two groups of 17 temporary, stand-alone seismic stations each. These were installed in the southeastern Cape Karoo region, near the town of Jansenville. Here we present data on the retrieval and coherency of Rayleigh surface waves extracted from the vertical component recordings. We reconstruct and show, for the first time in the southeastern Cape Karoo, estimates of Green's function from cross-correlating ambient noise data between stations pairs, which can be successfully used to image the subsurface. The stacked cross-correlations between all station pairs show clear arrivals of the Rayleigh surface waves. The group velocities of the Rayleigh waves in the 3 to 7 seconds period range were picked and inverted to compute the 2-D group velocity maps. The resulting 2-D group velocity maps at different periods resulted in a group velocity model from approximately 2 to 7 km depth, which shows a high velocity anomaly in the north of the study area, most likely imaging the denser, thick sedimentary basin of the Karoo (Carboniferous-Permian). To the south, the low velocity anomaly could correspond to the overlying Jurassic-Cretaceous sedimentary sequences of the younger Algoa Basin (Uitenhage Group).