Thermal desorption spectroscopy (TDS) is a highly sensitive and widely used method to directly measure the total hydrogen concentration and indirectly assess the hydrogen trapping sites and mechanisms from the spectra features in steels. Thus, there is a need to investigate the influence of sample location from a 5 mm plate of hot-rolled heat-resistant structural steel on TDS spectra. Via a new highly sensitive and specific correlation coefficient (microToH), the TDS results are correlated with low-angle grain boundaries volume fraction, grain size with different misorientation thresholds, microhardness, and geometrically necessary dislocations at different densities. The results indicate that sample location influences 133 % and 62 % in the hydrogen desorption of peak 1 (at 515 K), and total hydrogen concentration via the influence of peak 2 (at 634 K), respectively. Thus, sample location needs to be considered as one relevant aspect in a research plan based on TDS analysis. The influence on peak 3 (at 762 K) was found to be negligible as it is related to the first exothermic peak of the heating cycle, associated with carbide precipitation phenomena. The individual grain analysis performed with high-resolution adaptive DMM and GND maps emphasises the accumulation of the deformation in the ferritic domain at the vicinity of the pearlite structures.