Microbial diversity in surface waters was examined along a~ 15400 km transect of the Pacific Ocean from 70 N to 68 S latitude between late August and early November 2003. Comparative microbial diversity was determined using terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR amplified 16S and 18S rDNA restriction digested with CfoI and MspI. Bacterial numbers and total chlorophyll were greatest at higher latitudes in both hemispheres, with a smaller peak in equatorial waters. Flow cytometry analysis indicated a strong peak in Prochlorococcus from approximately 30 N to 30 S. Richness at each station was relatively low, with~ 11 prokaryotic peaks per sample and~ 12 eukaryotic peaks per community. Overall, prokaryotic populations appeared more diverse, with 181 total terminal restriction fragments (T-RFs) generated, while eukaryotic populations produced a total of 135 T-RFs. Prokaryotic and eukaryotic similarity dendrograms revealed 4 distinct cluster groups relating to regions sub-Arctic/Arctic, temperate, tropical and sub-Antarctic/Antarctic. T-RFLP patterns suggest that microbial communities may be influenced by ambient water temperature, with mid-latitudinal and equatorial communities more similar in composition to each other than to cold water communities. Global distribution of prokaryotic communities revealed an average inter-group similarity of~ 52%, while eukaryotic communities showed~ 51% similarity, implying that Pacific planktonic communities appear to be fairly homogenous in composition. Several T-RFs were ubiquitously distributed and contributed significantly to each cluster group, while several T-RFs were observed to be endemic to particular oceanic regions. Within-group similarities of> 70% were attributed to 12–14 T-RFs and 8–11 T-RFs in prokaryotic and eukaryotic profiles, respectively, suggesting that a small number of phylogenetic groups were responsible for each cluster group.