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Abstract Background Southeast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos. Methods Phlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions. Results A total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29). Conclusions This study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas. Graphical abstract