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Salmonella enterica infections continue to be a significant burden on public health worldwide. The ability of S. enterica to produce hydrogen sulfide (H2S) is an important phenotypic characteristic used to screen and identify Salmonella with selective medium; however, H2S-negative Salmonella have recently emerged. In this study, the H2S phenotype of Salmonella isolates was confirmed, and the selected isolates were subjected to antimicrobial susceptibility testing and molecular identification by multilocus sequence typing, pulsed-field gel electrophoresis, and clustered regularly interspaced short palindromic repeat (CRISPR) analysis. The phs genetic operon was also analyzed. A total of 160 S. enterica serovar Aberdeen isolates were detected between 2005 and 2013 in China. Of them, seven non-H2S-producing isolates were detected. Notably, four samples yielded four pairs of isolates with different H2S phenotypes, simultaneously. The data demonstrated that H2S-negative isolates were genetically closely related to H2S-positive isolates. Three new spacers (Abe1, Abe2, and Abe3) were identified in CRISPR locus 1 in four pairs of isolates with different H2S phenotypes from the same samples. Sequence analysis revealed a new nonsense mutation at position 208 in the phsA gene of all non-H2S-producing isolates. Additionally, we describe a new screening procedure to avoid H2S-negative Salmonella, which would normally be overlooked during laboratory and hospital screening. The prevalence of this pathogen may be underestimated; therefore, it is important to focus on improving surveillance of this organism to control its spread. [ABSTRACT FROM AUTHOR]