Argonaute (AGO) proteins are essential catalytic components of the RNA‐induced silencing complex and play central roles in RNA interference. Using a combination of bioinformatics and rapid amplification of cDNA ends (RACE) methods, putative AGO subfamily members, ls‐AGO1 and ls‐AGO2, were cloned and characterized from the small brown planthopper, Laodelphax striatellus. The open reading frame (ORF) of ls‐AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, and ls‐AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. The expected conserved PAZ and PIWI domains, and the conserved Asp–Asp–His (DDH) catalytic triad motif in the PIWI domain were observed in both ls‐AGO1 and ls‐AGO2. Reverse transcription‐qPCR (RT‐qPCR) results showed that both ls‐AGO1 and ls‐AGO2 were expressed in all developmental stages of L. striatellus with highest mRNA abundance in eggs. Expression of ls‐AGO1 and ls‐AGO2 was significantly decreased in adult insects in response to acquisition of rice black‐streaked dwarf virus by second instar nymphs. mRNA expression of ls‐AGO1 was significantly downregulated in response to low and high temperatures, but expression of ls‐AGO2 was only affected by low temperature. ls‐AGO1 and ls‐AGO2 were initially downregulated when insects were transferred from rice to maize and to the wild grass Brachypodium distachyon, but expression showed partial or complete recovery 7 days after transfer. These results document that AGO subfamily members of L. striatellus are ubiquitously expressed at different developmental stages and respond to various stresses. Thus, AGO subfamily may act in regulating the stress–response of L. striatellus by controlling related gene expression.