Genetic linkage maps in plants have been constructed by evaluating segregating populations derived from crosses between contrasting parents for traits of interest. Molecular markers are widely used as tools in this process and for effectiveness of genetic mapping they have to be highly polymorphic. The objective of this study was to evaluate polymorphism between contrasting parents for soybean oil content (Tucunaré and Line 69) which were used for the synthesis of a mapping population QTLs (Quantitative Trait Loci) for this trait, using 172 microsatellite markers or SSR (Simple Sequence Repeats). SSR polymorphisms were revealed by PCR amplifications that were carried out in a 25 μl volume containing 300 ng of template DNA, 10 μM of each forward and reverse primer, 0.2 mM of dNTP, 4.0 mM of MgCl₂, 1X Buffer (10 mM Tris-HCl pH 8, 50mM KCl) and 1 U Taq DNA polymerase. After an initial denaturing step of 7 minutes at 94 °C, the PCR amplification was performed in 32 cycles of 1 minute at 94 °C, 1 minute of annealing at the specific temperature of each primer and 2 minutes of extension at 72 °C, followed by a final extension at 72 °C for 7 minutes and then kept at 4 °C. Amplification products were separated by electrophoresis in 3% agarose gel and stained with ethidium bromide. SSR markers were genotyped considering presence (1) or absence (0) of a determined DNA fragment for different samples. Of the 172 characterized SSRs, 149 (87%) generated high quality of banding patterns being possible to detect 224 alleles, where the number of alleles amplified per locus ranged of one to two. 74 SSRs were monomorphic while 75 SSRs detected polymorphism between the parents, indicating that the population mapping obtained by these genotypes may be effective to construct a genetic linkage map for seed oil content in soybean.