Objective To analyze the pathogen of 3 kala-azar infants with negative bone marrow smear by metagenomics next-generation sequencing (mNGS), and to explore the clinical application value of mNGS. Methods Clinical data of 3 pediatric cases of infant kala-azar with negative bone marrow smear collected in PICU from April 2021 to February 2022 were restrospectively analyzed. Results Two of the three infants were girls and one was a boy, all under the age of three. All had fever, negative bone marrow smears, peripheral blood mNGS tests suggestive of Leishmania protozoa infection, and no other pathogenic infections were observed; all were cured after antimony gluconate treatment. Conclusions Early diagnosis of infant kala-azar with negative bone marrow smear is difficult. mNGS can provide rapid and accurate pathogenetic diagnostic support and aid in precise diagnosis and treatment for clinical infectious diseases, particularly complicated and critical infectious diseases whose pathogens cannot be determined by routine testing.