Rubella virus (RV) continues to circulate in the Indian population, as rubella vaccination is not widespread. Congenital cataract due to maternal rubella constitutes a significant cause of visual loss among Indian infants. The aim of this study was to isolate and characterize RV from a range of specimens obtained from infants with serologically confirmed congenital rubella infection (CRI). Vero and RK13 cell lines were used for RV isolation, and RV strains were identified by indirect immunofluorescence (IFA), real-time and nested PCR assays. In total, 30 specimens from 17 infants were investigated. Twenty-two samples including two oral fluids (OF), four throat swabs and 16 lenses were simultaneously cultured in two cell lines. A total of 26 viruses were successfully isolated, 14 in Vero cells and 12 in RK13 cells. All 26 isolates were confirmed by PCR and 21/26 by IFA. For the first time, RV has been isolated from an oral fluid specimen. Higher viral loads were detected in Vero cell cultures, but a cytopathic effect was observed only in the RK13 cells when inoculated with lens material.  In addition, two infants (case 9 and 12) were confirmed by PCR assay direct from their lens specimens. The generated sequences of the 739 nucleotide of E1 gene confirmed the presence of RV genotype 2B in South India. The point mutations observed in these RV strains may reflect geographic and temporal differences, adaptation to cell culture or the long period of incubation in CRS patients.