The main factors influencing the spatiotemporal variability of urban climate are quite widely recognized, including, for example, the thermal properties of materials used for surfaces and buildings, the mass, height and layout of the buildings themselves and patterns of land use. However, the roles played by particular factors vary from city to city with respect to differences in geographical location, overall size, number of inhabitants and more. In urban climatology, the concept of “local climate zones” (LCZs) has emerged over the past decade to address this heterogeneity. In this contribution, a new GIS-based method is used for LCZ delimitation in Prague and Brno, the two largest cities in the Czech Republic, while land surface temperatures (LSTs) derived from LANDSAT and ASTER satellite data are employed for exploring the extent to which LCZ classes discriminate with respect to LSTs. It has been suggested that correctly-delineated LCZs should demonstrate the features typical of LST variability, and thus, typical surface temperatures should differ significantly among most LCZs. Zones representing heavy industry (LCZ 10), dense low-rise buildings (LCZ 3) and compact mid-rise buildings (LCZ 2) were identified as the warmest in both cities, while bodies of water (LCZ G) and densely-forested areas (LCZ A) made up the coolest zones. ANOVA and subsequent multiple comparison tests demonstrated that significant temperature differences between the various LCZs prevail. The results of testing were similar for both study areas (89.3% and 91.7% significant LST differences for Brno and Prague, respectively). LSTs computed from LANDSAT differentiated better between LCZs, compared with ASTER. LCZ 8 (large low-rise buildings), LCZ 10 (heavy industry) and LCZ D (low plants) are well-differentiated zones in terms of their surface temperatures. In contrast, LCZ 2 (compact mid-rise), LCZ 4 (open high-rise) and LCZ 9 (sparsely built-up) are less distinguishable in both areas analyzed. Factors such as seasonality and thermal anisotropy remain a challenge for future research into LST differences.