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Salmonella, one of the major causes of foodborne infections, can cause bacterial foodborne illness. We investigated the serotype distribution, multidrug resistance (MDR), and β-lactamase resistance genes of human Salmonella isolates collected from clinical specimens in Guizhou, China, between 2013 and 2018. A total of 363 Salmonella isolates were collected from clinical specimens at 17 surveillance hospitals. Twenty-four serotypes were identified by sliding agglutination test. S. Enteritidis (33.9%), Salmonella 4,[5],12:i:- (24.0%), S. Typhimurium (16.3%), S. London (6.3%), and S. Derby (3.9%) were the top five serotypes. In 2018, the most common serotype changed from S. Enteritidis to S. Typhimurium. Among the 363 Salmonella isolates, 97.5% of isolates were resistant to at least one class of antimicrobial agents. For cephalosporins, ceftriaxone had the highest resistance rate of 10.5%, and cefepime and cefoxitin were 8.0% and 2.2%, respectively. Three hundred and one (82.9%) Salmonella isolates showed MDR. Salmonella 4,[5],12:i:- had the highest MDR rate with 94.2%, followed by S. London (91.3%) and S. Typhimurium (88.1%). Multidrug resistance rates of Salmonella isolates in Guizhou from 2013 to 2017 increased from 75.8% to 86.7%. Sixteen isolates (4.4%) showed extensive drug resistance. One hundred thirty-four antimicrobial resistance patterns were found. Two hundred and forty-one (66.4%) isolates carried at least one β-lactamase resistance gene. The blaTEM gene (61.2%) was the most prevalent resistant gene in all Salmonella isolates, followed by the blaCTX-M gene (6.1%) and blaOXA-1 gene (4.1%). Our findings showed that the MDR rate of Salmonella isolates from Guizhou province increased year by year. Therefore, systematic and long-term surveillance on MDR Salmonella isolates from clinical patients should be further strengthened. [ABSTRACT FROM AUTHOR]