TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that can be employed to generate allelic series of induced mutations in targeted genes for functional analyses. To date, TILLING resources in Arabidopsis thaliana are only available in accessions Columbia and Landsberg erecta. Here, we extended the Arabidopsis TILLING resources by developing a new population of ethyl methanesulfonate (EMS)-induced mutant lines in another commonly used A. thaliana accession C24. A permanent collection of 3,509 independent EMS mutagenized M2 lines was developed in A. thaliana accession C24, and designated C24TILL. Using the TILLING method to search C24TILL for mutations in four selected genes identified a total of 73 mutations, comprising 69.6% missense, 29.0% sense, and 1.4% nonsense mutations. Consistent with the propensity of EMS to induce guanine alkylation, 98.4% of the observed mutations were G/C to A/T transitions. Based on the mutations identified in the four target genes, the overall mutation density in the C24TILL collection was estimated to be 1/345 kb. TILLING the DUO POLLEN 1 (DUO1) gene from the C24TILL collection identified a truncation mutation leading to a deficiency in sperm cell differentiation. Taken together, a new TILLING resource, the C24TILL collection, was generated for A. thaliana accession C24. The C24TILL collection provides an allelic series of induced point mutations that will serve as a useful alternative reverse genetic resource for functional genetic studies in A. thaliana.