Mitochondrial dysfunction has been associated with several human pathologies, including cancer, aging and neurodegenerative diseases. Thus, the availability of selective fluorescent probes for mitochondria could play an important role in the future for monitoring cellular function and disease progression. In this work, we have studied how the photophysical properties and subcellular accumulation of nonconventional coumarin-based COUPY fluorophores can be fine-tuned through replacement of the para-pyridinium moiety with several heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties for bioimaging applications, including emission in the far-red to NIR region, large Stokes' shifts and high photostability.Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria compared with the parent COUPY fluorophores.Overall, these results provide useful insights for the development of novel mitochondriatargeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-pyrimidinium counterparts.