Background: Thrombocytopenia has a variety of different etiologies, both acquired and hereditary. Inherited thrombocytopenia may be associated with other symptoms (syndromic forms) or may be strictly isolated. To date, only about half of all the familial forms of thrombocytopenia have been accounted for in terms of well-defined genetic abnormalities. However, data are limited on the nature and frequency of the underlying causative genetic variants in individuals with mild isolated nonsyndromic thrombocytopenia. Study design and methods: Thirteen known or candidate genes for isolated thrombocytopenia were included in a gene panel analysis in which targeted next-generation sequencing was performed on 448 French blood donors with mild isolated nonsyndromic thrombocytopenia. Results: A total of 68 rare variants, including missense, splice site, frameshift, nonsense, and in-frame variants (all heterozygous) were identified in 11 of the 13 genes screened. Twenty-nine percent (N = 20) of the variants detected were absent from both the French Exome Project and gnomAD exome databases. Using stringent criteria and an unbiased approach, we classified seven predicted loss-of-function variants (three in ITGA2B and four in TUBB1) and four missense variants (one in GP1BA, two in ITGB3 and one in ACTN1) as being pathogenic or likely pathogenic. Altogether, they were found in 13 members (approx. 3%) of our studied cohort. Conclusion: We present the results of gene panel sequencing of known and candidate thrombocytopenia genes in mild isolated nonsyndromic thrombocytopenia. Pathogenic and likely pathogenic variants in five known thrombocytopenia genes were identified, accounting for approximately 3% of individuals with the condition.