BackgroundIlex pubescensis an important traditional Chinese medicinal plant with many naturally occurring compounds and multiple pharmacological effects. However, the lack of reference genomic information has led to tardiness in molecular biology research and breeding programs of this plant.ResultsTo obtain knowledge on the genomic information ofI. pubescens,a genome survey was performed for the first time by next generation sequencing (NGS) together with genome size estimation using flow cytometry. The whole genome survey ofI. pubescensgenerated 46.472 Gb of sequence data with approximately 82.2 × coverage. K-mer analysis indicated thatI. pubescenshas a small genome of approximately 553 Mb with 1.93% heterozygosity rate and 39.1% repeat rate. Meanwhile, the genome size was estimated to be 722 Mb using flow cytometry, which was possibly more precise for assessment of genome size than k-mer analysis. A total of 45.842 Gb clean reads were assembled into 808,938 scaffolds with a relatively short N50 of 760 bp. The average guanine and cytosine (GC) content was 37.52%. In total, 197,429 microsatellite motifs were detected with a frequency of 2.8 kb, among which mononucleotide motifs were the most abundant (up to 62.47% of the total microsatellite motifs), followed by dinucleotide and trinucleotide motifs.ConclusionIn summary, the genome ofI. pubescensis small but complex with a high level of heterozygosity. Even though not successfully applied for estimation of genome size due to its complex genome, the survey sequences will help to design whole genome sequencing strategies and provide genetic information support for resource protection, genetic diversity analysis, genetic improvement and artificial breeding ofI. pubescens.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09359-5.