Reverse complementary DNA sequences – sequences that are inadvertently given backwards with all purines and pyrimidines transposed – can affect sequence analysis detrimentally unless taken into account. We present an open-source, high-throughput software tool –v-revcomp (http://www.cmde.science.ubc.ca/mohn/software.html) – to detect and reorient reverse complementary entries of the small-subunit rRNA (16S) gene from sequencing datasets, particularly from environmental sources. The software supports sequence lengths ranging from full length down to the short reads that are characteristic of next-generation sequencing technologies. We evaluated the reliability of v-revcomp by screening all 406 781 16S sequences deposited in release 102 of the curated SILVA database and demonstrated that the tool has a detection accuracy of virtually 100%. We subsequently used v-revcomp to analyse 1 171 646 16S sequences deposited in the International Nucleotide Sequence Databases and found that about 1% of these user-submitted sequences were reverse complementary. In addition, a nontrivial proportion of the entries were otherwise anomalous, including reverse complementary chimeras, sequences associated with wrong taxa, nonribosomal genes, sequences of poor quality or otherwise erroneous sequences without a reasonable match to any other entry in the database. Thus, v-revcomp is highly efficient in detecting and reorienting reverse complementary 16S sequences of almost any length and can be used to detect various sequence anomalies.