Distribution of serotypes and alpha-like surface protein (Alp) of Streptococcus agalactiae (Group B Streptococci - GBS) vary with geographical region, ethnic origin and the virulence of clinical isolates. Demonstration of different genotypes based on surface protein genes improves the potential of GBS subtyping, is essential in research on new vaccines against invasive neonatal infections and may be useful in epidemiological studies. The molecular characterization of protein gene profile of GBS isolates was the main aim of this study. We evaluated the applicability of multiplex PCR for the identification of GBS protein genes from Alp family, such as: epsilon, bca, rib, alp2, alp3, alp4 and evaluated presence of these genes in the group of GBS isolates originating from vaginal or rectal carriage in pregnant women. For statistical analysis the G2 (Likelihood ratio) test was used. P values of < 0.05 were considered significant. The surface protein genes were found in all investigated strains. The epsilon gene dominated (27%) in GBS isolates originating from healthy pregnant women. The other genes were detected with the following frequency: rib (21%), alp2 (21%), bca (17%) and alp3 (14%). In the analyzed population, GBS strains with alp4 gene were not found. A statistically significant relationship between surface protein genes and capsular polysaccharides was demonstrated (p < 0.0001). The results of our study show immense diagnostic usefulness of multiplex PCR for identification of genes encoding GBS surface proteins from Alp family.