Development of microsatellite markers for sunflower (Helianthus annuus L.) was performed to estimate their frequency, nature (structure), levels of polymorphism, usefulness for genotype identification, and calculation of genetic relationships between inbred lines representing the species diversity. Isolation was performed from a small-insert genomic library followed by hybridization screening using oligonucleotide probes containing different nucleotide arrays. In this work, 503 unique microsatellite clones were sequenced and 271 PCR primer sequences bordering the microsatellite repeat were designed. For polymorphism assessment, 16 H. annuus germplasm accessions were checked and 170 of the primers tested were shown to be polymorphic for the selected lines. The polymorphic microsatellites produced an average of 3.5 alleles/locus and an average polymorphism information content (PIC) of 0.55. The most frequently found motifs within polymorphic simple-sequence repeats (SSRs) were: (GA)n, (GT)n, (AT)n, followed by trinucleotides (ATT)n, (TGG)n, and (ATC)n, and the tetranucleotide (CATA)n. Most of the 170 SSRs obtained showed important differences in the 16 reference inbred lines used for their characterization. In this work, 20 of the most informative SSRs destined to sunflower genotyping and legal fingerprinting purposes are fully described.