The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species' genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine.