Abstract Background Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. Methods CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. Results CASAL had ∼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1–100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. Conclusions CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.