The RNA-templated extension of oligoribonucleotides by nucleotides produces either a 3',5' or a 2',5'-phosphodiester. Nature controls the regioselectivity during RNA chain growth with polymerases, but enzyme-free versions of genetic copying have modest specificity. Thus far, enzymatic degradation of products, combined with chromatography or electrophoresis, has been the preferred mode of detecting 2',5'-diesters produced in enzyme-free reactions. This approach hinges on the substrate specificity of nucleases, and is not suitable for in situ monitoring. Here we report how 1 H NMR spectroscopy can be used to detect the extension of self-templating RNA hairpins and that this reveals the regioisomeric nature of the newly formed phosphodiesters. We studied several modes of activating nucleotides, including imidazolides, a pyridinium phosphate, an active ester, and in situ activation with carbodiimide and organocatalyst. Conversion into the desired extension product ranged from 20 to 90 %, depending on the leaving group. Integration of the resonances of H1' protons of riboses and H5 protons of pyrimidines gave regioselectivities ranging from 40:60 to 85:15 (3',5' to 2',5' diester), but no simple correlation between 3',5' selectivity and yield. Our results show how monitoring with a high-resolution technique sheds a new light on a process that may have played an important role during the emergence of life.