Classically Salmonella enterica serovar Typhi (S. Typhi) is associated with typhoid, a major health problem in developing countries. However, in recent years Salmonella enterica serovar Paratyphi A (S. Paratyphi A) and Vi negative variants of S. Typhi have emerged rapidly. For development of multiple protein-polysaccharide conjugates with potential to be used as vaccines, we needed to have a first hand knowledge of local distribution of these pathogens. For this purpose, we developed a nested multiplex PCR targeting 5 different genes for differential diagnosis of typhoidal pathogens which has been optimized to be directly applicable on clinical blood samples. Out of 42 multiplex PCR positive blood samples, 26, 9, and 2 were Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A, respectively and 5 patients were found to have mixed infection. Seventeen patients grew Salmonella from blood culture and the remaining 25 were positive in the Salmonella specific PCR. Tests with several common pathogens confirmed the specificity of the assay. We conclude that the proposed multiplex PCR is rapid, sensitive and specific for the diagnosis of typhoidal pathogens directly from blood samples. Detection of Vi negative strains of S. Typhi and S. Paratyphi A among typhoidal patients suggested the need to work on vaccines which are not based on Vi polysaccharide only. Therefore, O-specific polysaccharides (OSP) purified from lipopolysaccharides (LPS) of S. Typhi and S. Paratyphi A were conjugated with diphtheria toxoid (DT) using adipic acid dihydrazide (ADH) as linker, and evaluated for immunogenicity in mice. Use of DT as carrier protein for Salmonellae has not been reported before. S. Typhi OSP-AH-DT conjugate 1 and 2 elicited significantly higher IgG anti-LPS ELISA titer (P = 0.0241 and 0.0245 respectively) than polysaccharide alone. The injection schedule-B (three injections with 4-weeks interval) was found better than schedule-A (three injections with 2-weeks interval). The conjugate of S. Paratyphi A OSP with DT without linker molecule did not elicit sufficient immune response to be used as conjugate vaccine candidate while antibody response against S. Paratyphi A OSP-A H-DT c onjugate was found significantly higher (P = 0.0446) than polysaccharide alone.