This study deals with the spatial pattern of university spin-off companies from universities in peripheral regions of the Netherlands, to understand the economic impact that these companies have on the regional economy. The study tracks the spin-offs over time and shows their development and migration pattern. Most university spin-offs are located close to their parent university, but many of them are also attracted by large urban agglomerations in economic core areas. When looking at the size of spin-offs, in terms of number of workplaces, only in a handful of larger cities, the direct employment in spin-offs reaches levels of 3 to 5% of the total employment, a level that could be seen as moderately impactful and, in such cities, university spin-offs could indeed be called “game changers” in economic development. The indirect economic impact of the spin-off businesses is difficult to pinpoint. The goal of spin-offs is to develop newly developed university knowledge into practical business solutions, but it is difficult to measure the impact that these new technologies have on other businesses. As university spin-off companies are a highly heterogeneous group of companies, the study breaks down the group of spin-offs into four subgroups, of which subgroup 1 is the most innovative and subgroup 4 the least innovative. Statistical analyses show that those subgroups of university spin-offs significantly differ in terms of company development and spatially behave in a different way, with the most innovative spin-offs being most likely located near the parent university and the least innovative spin-offs being most likely located in large urban agglomerations. The study also compares spin-offs from research universities with those of applied science universities. Spin-offs from these higher education institutions significantly differ from one another in spatial pattern and growth. Applied science university spin-offs are more spread out through the region and less concentrated within the larger cities of regions, therefore likely contributing more to the development of more rural areas within regions.