Listeria monocytogenes has been isolated from catfish and various non-catfish seafoods. Despite progress that was made to understand the relationship between L. monocytogenes isolated from seafood and humans, no study has emphasized the genetic relatedness between catfish and non-catfish seafood and human isolates. The objectives of this study were to (1) investigate the genetic relationship between L. monocytogenes isolates from catfish, non-catfish seafood and humans using pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic element-based PCR (REP-PCR) linking with serotypes; and (2) compare the distinct capabilities of these methods. The genetic relationship among 36 catfish, 35 non-catfish seafood, and 57 human isolates were analyzed using Bionumerics software. Among a total of 128 isolates, only two subtypes by PFGE with ApaI-digestion, one subtype by PFGE with AscI-digestion and three subtypes by REP-PCR were shared by non-catfish seafood or catfish with human isolates. Although most of the non-catfish seafood and catfish isolates were genetically distinct from human isolates, the human isolates used in this study may not be truly representative of all clinical isolates. Serotype 4b was dominant in human isolates, whereas serotypes 3b, 1/2a, and 1/2b and serotypes 4b, 1/2a and 1/2b were commonly found in catfish and non-catfish seafood, respectively. Furthermore, 97, 87 and 94 subtypes of L. monocytogenes were revealed using the ApaI-digestion, the AscI-digestion, and the REP-PCR, respectively. Their respective discriminatory indexes were 0.994, 0.990 and 0.994. Distinct genetic groups based on the difference of flagella antigens were observed in all three methods. The study suggests that the REP-PCR possesses a similar discriminatory ability as the PFGE for subtyping L. monocytogenes. Therefore, the REP-PCR that is rapid and less expensive could be considered as an alternative method for subtyping L. monocytogenes.