© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. Studying how bacterial strains diverge over space and time and how divergence leads to ecotype formation is important for understanding structure and dynamics of environmental communities. Here we assess the ecological speciation and temporal dynamics of a collection of Shewanella baltica strains from the redox transition zone of the central Baltic Sea, sampled at three time points over a course of 12 years, with a subcollection containing 46 strains subjected to detailed genetic and physiological characterization. Nine clades were consistently recovered by three different genotyping approaches: gyrB gene sequencing, multilocus sequence typing (MLST) and whole genome clustering of data from comparative genomic hybridization, and indicated specialization according to nutrient availability, particle association and temporal distribution. Genomic analysis suggested higher intra- than inter-clade recombination that might result from niche partitioning. Substantial heterogeneity in carbon utilization and respiratory capabilities suggested rapid diversification within the same 'named' species and physical habitat and showed consistency with genetic relatedness. At least two major ecotypes, represented by MLST clades A and E, were proposed based on genetic, ecological and physiological distinctiveness. This study suggests that genetic analysis in conjunction with phenotypic evaluation can provide better understanding of the ecological framework and evolutionary trajectories of microbial species.