Objective To analyze the laboratory detection results of leishmaniasis in Henan Province from 2013 to 2023, so as to provide data support for leishmaniasis control in Henan Province. Methods Suspected leishmaniasis cases’ anticoagulant whole blood samples, bone marrow smear samples and injured tissue samples were collected from the laboratory of Institute of Parasitic Disease Prevention and Control Institute Henan Center for Disease Control and Prevention from 2013 to 2023. All samples were collected from individuals retested positive for rK39 immunochromatographic test strips from all city-level disease control and prevention centers in Henan Province from 2013 to 2023, and cases’ characteristics were retrieved from the Infectious Diseases Surveillance System of the Information System of Chinese Center for Disease Control and Prevention. All samples were detected using microscopic examination, rK39 strips and PCR assay, and bidirectional sequencing was performed following PCR amplification. Successfully sequenced gene sequences were spliced and assembled using the software ChromasPro 1.5 and aligned using the BLAST tool. All statistical analyses were conducted using the statistical software SPSS 21.0. Differences of the positive rates of leishmaniasis by different assays were tested for statistical significance with Fisher’s exact test. Results A total of 186 samples were collected, originating from 137 suspected cases from 2013 to 2023, with the three highest numbers of cases from Anyang City (51, 37.23%), Zhengzhou City (44, 32.12%) and Luoyang City (31, 22.63%), and the highest number in 2023 (37.23%, 51/137). There were 94 cases from men and 43 from women, with a male to female ratio of 2.19:1, and leishmaniasis cases were predominantly at ages of 0-< 10 years (41, 29.93%) and 60-< 70 years (24, 17.52%). Microscopic examinations detected a positive rate of 62.00% in 50 bone marrow smears (31/50), and PCR assay and rK39 strips revealed positive rates of 66.92% (89/133) and 75.19% (100/133) in 133 anticoagulant whole blood samples, while PCR assay identified 2 positives in 3 injured tissue samples. A total of 92 cases were definitely diagnosed as leishmaniasis in 137 cases by means of microscopic examinations combined with PCR assay, with a positive rate of 67.15%. There was no significant difference in the positive rate of leishmaniasis between PCR assay and microscopic examinations (Fisher’s exact test, P > 0.05), and there were significant differences between microscopic examinations and rK39 strips and between PCR assay and rK39 strips (Fisher’s exact test, P < 0.05). The diagnostic sensitivity and specificity were 98.93% and 100% for PCR assay, 94.49% and 66.68% for rK39 strips and 76.92% and 90.91% for microscopic examinations, respectively. Seventy three out of 91 PCR assay-positive samples were successfully sequenced, and BLAST alignment revealed Leishmania infantum in 71 samples and L. major in 2 samples. Conclusion L. infantum remains predominant Leishmania species in Henan Province, with leishmaniasis cases primarily found from three cities of Anyang, Zhengzhou, and Luoyan. Intensified leishmaniasis control and improved laboratory detection capability are recommended.