The molecular diversity of marine picocyanobacterial populations, an important component of phytoplankton communities, is better characterized using high-resolution marker genes than the 16S rRNA gene as they have greater sequence divergence to differentiate between closely related picocyanobacteria groups. Although speciﬁc ribosomal primers have been developed, another general disadvantage of bacterial ribosome-based diversity analyses is the variable number of rRNA gene copies. To overcome these issues, the single-copy petB gene, encoding the cytochrome b6 subunit of the cytochrome b6f complex, has been used as a high-resolution marker gene to characterize Synechococcus diversity. We have designed new primers targeting the petB gene and proposed a nested PCR method (termed Ong_2022) for metabarcoding of marine Synechococcus populations obtained by ﬂow cytometry cell sorting. We evaluated the speciﬁcity and sensitivity of Ong_2022 against the standard ampliﬁcation protocol (termed Mazard_2012) using ﬁltered seawater samples. The Ong_2022 approach was also tested on ﬂow cytometry-sorted Synechococcus populations. Samples (ﬁltered and sorted) were obtained in the Southwest Paciﬁc Ocean, from subtropical (ST) and subantarctic (SA) water masses. The two PCR approaches using ﬁltered samples recovered the same dominant subclades, Ia, Ib, IVa, and IVb, with small differences in relative abundance across the distinct samples. For example, subclade IVa was dominant in ST samples with the Mazard_2012 approach, while the same samples processed with Ong_2022 showed similar contributions of subclades IVa and Ib to the total community. The Ong_2022 approach generally captured a higher genetic diversity of Synechococcus subcluster 5.1 than the Mazard_2012 approach while having a lower proportion of incorrectly assigned amplicon sequence variants (ASVs). All ﬂow cytometry-sorted Synechococcus samples could be ampliﬁed only by our nested approach. The taxonomic diversity obtained with our primers on both sample types was in agreement with the clade distribution observed by previous studies that applied other marker genes or PCR-free metagenomic approaches under similar environmental conditions.