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• The first Hemerocallis core collection from 155 accessions was constructed based on both the phenotypic data and EST-SSR markers of 33 traits and a stepwise clustering preferred sampling strategy. • The 32 accessions in the core collection captured majority of molecular, extensive phenotypic diversity for yield and other horticulturally important traits in the original population, which isuseful for night lily improvement. • Clustering analysis of the core collection classified the 32 accessions into two major groups: night lily and daylily, with the daylily being more ancestral and the night lily having a narrower genetic basis. The night lily, Hemerocalliscitrina Baroni, is an economically important specialty crop widely grown in East Asia countries. It is mainly consumed as a vegetable for the dried flower buds, and is also for medicinal use. Heme-rocallis sp. is becoming a model plant for understanding of several interesting biological phenomena in biology, such as self-incompatibility, circadian rhythm and programmed cell death. Understanding the genetic diversity and phylogenetic relationships of the Hemerocallis germplasm is essential for efficient use and improvement of the night lily. In this study, from 155 Hemerocallis accessions, a core collection was constructed based on EST-SSR markers, phenotypic data from 33 traits, and a stepwise clustering preferred sampling strategy. The 32 accessions in the core collection captured majority of molecular and morphological diversities in the original population. While the molecular data and morphological data provided equal power in assessing the genetic diversity of the core collection, the pollen morphology has its limitation in the study of the genetic relationship within Hemero-callis. Clustering analysis of the core collection classified the 32 accessions into two major groups: night lily and daylily, with the daylily being more ancestral and the night lily having a narrower genetic basis. There is exten-sive phenotypic diversity for yield and other horticulturally important traits in the Hemerocallis core collection that is useful for night lily improvement. [Display omitted] [ABSTRACT FROM AUTHOR]