Isothermal nucleic acid amplification techniques are promising alternatives to polymerase chain reaction (PCR) for amplifying and detecting nucleic acids under resource-limited conditions. While many isothermal amplification strategies, such as recombinase polymerase amplification (RPA), offer comparable sensitivity to PCR, they often lack the specificity and robustness for discriminating single nucleotide variants (SNVs), mainly due to the uncontrolled production of massive amplicons. Herein, we introduce a mismatch-guided DNA assembly (MGDA) approach capable of discriminating SNVs in the presence of high concentrations of wild-type (WT) interferences. We show that an optimal MGDA design can effectively suppress interfering signals from WT while maintaining high detection signals for the targeted SNV. A further introduction of a competitive sink probe allowed the detection of challenging SNVs, such as those containing G-T wobbles, with high sensitivity and specificity. Because it is highly specific and tolerant to massively produced interfering amplicons during isothermal nucleic acid amplification, we integrated MGDA with RPA for discriminating clinically relevant SNVs in point-of-care settings. We demonstrate that our RPA-MGDA is highly sensitive and specific, allowing the detection of as low as 1 aM SNVs with an allele frequency of 0.5%. We also evaluated the clinical potential of RPA-MGDA by analyzing epidermal growth factor receptor L858R mutations in tumor tissue samples collected from non-small-cell lung cancer patients (n = 44). A multiplexed RPA-MGDA assay was also developed for the simultaneous detection of pharmacogenetic mutations in buccal swab samples (n = 30).