Background: The prognosis of multiple myeloma (MM) patients is dependent on minimal residual disease (MRD) status after treatment. Multicolor flow cytometry (MFC) as a clinical MRD evaluation method has certain limitations regarding sample requirement, sensitivity and specificity. Next-generation sequencing (NGS)-based B cell receptor (BCR) clonality assessment has an edge in lymphoid clone MRD detection, with easier sample preparation and higher sensitivity. NGS-based BCR clonality assays were performed to evaluate curative effect and track MRD of MM patients, and its performance in testing samples with limited DNA contents was explored. Methods: In this retrospective study, newly diagnosed or relapsed MM patients with paired sample collected before and after initial treatment were included, and OriMIRACLE L NGS assay were used for BCR clonality detection in genomic DNA (gDNA) extracted from clinical residual marrow smear samples. The quantitative accuracy of NGS was compared to MFC. The concordance of MRD between MFC, NGS and clinical response assessment were analyzed. Results: From December 2016 to April 2022, 29 patients were included with a median age of 65 years old. The gDNA input of the sample collected before treatment was 100.0 ng, and the median DNA content of the sample collected after treatment was 1673.4 ±594.8 ng. The quantitative results of NGS and MFC were consistent in samples with a frequency of cancer cells of above 1x10^-4 among all nucleated cells. BCR clonality detection in 23 newly diagnosed and 6 relapsed cases showed a 100% sensitivity at diagnosis. For post-treatment monitoring, patients with positive MRD by both MFC and NGS (n = 18), the MRD results were consistent with clinical response assessment. One of the patients achieved complete response (CR), while the remaining patients were unable to achieved CR. The CR patient was relapsed after 134 days. The clinical responses of 11 cases with negative MRD by MFC and positive MRD by NGS were reviewed. There were 7 cases with CR, 3 cases with very good partial response or partial response, and one case with stable disease. Among those CR patients, except for one patient who was lost to follow-up, 4 patients who underwent continuous treatment were still CR and 2 patients who had treatment discontinued recurred at 67 and 134 days. Conclusions: OriMIRACLE L NGS assay showed the higher sensitivity and specificity of MRD evaluation than MFC. For patients with negative MRD by MFS and positive MRD by NGS, recurrence might occur in case of treatment discontinuation. It is recommended to consider carefully that whether maintenance treatment is required in such cases.