Abstract The elimination of Plasmodium vivax is challenged by dormant liver stages (hypnozoites) that can reactivate months after initial infection resulting in relapses that enhance transmission. Relapsing infections confound antimalarial clinical efficacy trials due to the inability to distinguish between recurrences arising from blood-stage treatment failure (recrudescence), reinfection or relapse. Genetic relatedness of paired parasite isolates, measured by identity-by-descent (IBD), can provide important information on whether individuals have had single or multiple mosquito inoculations, thus informing on recurrence origin. We developed a high-throughput amplicon sequencing assay comprising 93 multi-SNP (microhaplotype) P. vivax markers to determine IBD between P. vivax clinical isolates. The assay was evaluated in 659 independent infections from the Asia-Pacific and Horn of Africa, including 108 pairs of infections from a randomized controlled trial (RCT). A bioinformatics pipeline (vivaxGEN-MicroHaps) was established to support data processing. Simulations using paneljudge demonstrated low error in pairwise IBD estimation in all countries assessed (all RMSE <0.12) and IBD-based networks illustrated strong clustering by geography. IBD analysis in the RCT demonstrated a higher frequency of suspected relapses or recrudescence in patients treated with primaquine regimens (0.84) compared to those without primaquine (0.60). Our results demonstrate the potential to derive information on P. vivax IBD using amplicon sequencing, that informs policy-relevant treatment and transmission dynamics.