ABSTRACT Background Basophils are implicated in various diseases including allergies, but a comprehensive single‐cell characterization of human basophils has yet to be performed. Here, we aimed to generate a single‐cell omics‐based reference resource of circulating human basophils, integrating transcriptomic and large‐scale immunoprofiling data. We also sought to investigate basophil heterogeneity at the molecular level. Methods Circulating basophils were analyzed using cellular indexing of transcriptomes and epitopes by sequencing (CITE‐seq). Both short‐ and long‐read single‐cell RNA‐sequencing platforms were used to capture the transcriptomic data. Results CITE‐seq enabled accurate identification and profiling of side scatter low lineage − CCR3 + FcεRI + basophils. Short‐read single‐cell RNA‐sequencing data revealed two previously unresolved basophil populations, defined by 66 differentially expressed genes and reproducibly identified across donors. Despite the transcriptional differences, the populations displayed similar immunophenotypes based on more than 100 investigated cell surface markers. Long‐read single‐cell RNA‐sequencing analysis confirmed the existence of the two populations and provided further insights into their gene expression profiles. Conclusions We present a multimodal single‐cell resource that defines two novel transcriptionally distinct basophil populations. This resource, accessible through a user‐friendly web interface, constitutes a cellular and molecular reference map for future studies of basophils in health and disease.