Developing a clearer understanding of electron tunneling through molecules is a central challenge in molecular electronics. Here we demonstrate the use of mechanical stretching to distinguish orbital pathways that facilitate tunneling in molecular junctions. Our experiments employ junctions based on self-assembled monolayers (SAMs) of homologous alkanethiols (CnT) and oligophenylene thiols (OPTn), which serve as prototypical examples of σ-bonded and π-bonded backbones, respectively. Surprisingly, molecular conductances (G molecule ) for stretched CnT SAMs have exactly the same length dependence as unstretched CnT SAMs in which molecular length is tuned by the number of CH 2 repeat units, n. In contrast, OPTn SAMs exhibit a 10-fold-greater decrease in G molecule with molecular length for stretched versus unstretched cases. Experiment and theory show that these divergent results are explained by the dependence of the molecule-electrode electronic coupling δ on strain and the spatial extent of the principal orbital facilitating tunneling. In particular, differences in the strain sensitivity of δ versus the repeat-length (n) sensitivity can be used to distinguish tunneling via delocalized orbitals versus localized orbitals. Angstrom-level tuning of interelectrode separation thus provides a strategy for examining the relationship between orbital localization or delocalization and electronic coupling in molecular junctions and therefore for distinguishing tunneling pathways.