Five new Rhodobacter sphaeroides bacteriophages designated øR3-PR1-5 were isolated from water reservoirs at the Southwest region of Puerto Rico. The organic solvent susceptibility test showed that these phages lacked of external lipidic envelope. The øR3-PR bacteriophages were able to form plaques using R. sphaeroides as host only during aerobic conditions. Ultrastructural (Transmission Electron Microscopy; TEM) analysis revealed that all phages had polyhedral heads (90 × 120nm to 20 × 20nm) and a long tail (20 to 200nm). The TEM analysis of the isolates is similar to the bacteriophages previously described for the Myoviridae and Siphoviridae family. In the host specificity analysis all phages infected the restriction deficient mutant R. sphaeroides 7001, one infected R. sphaeroides strain 630 and 2.4.1. The isolated bacteriophages were double-stranded DNA with genome sizes that range from 33–56 kbp. Restriction analysis was also performed using BglII, ClaI, EcoRI, KpnI, HindIII, SmaI and SalI endonucleases. All the data collected in this study confirm that the øR3-PR1-5 bacteriophages are different among themselves and from the R. sphaeroides bacteriophages previously reported in the literature.