Effective laboratory methods for identifying avian influenza virus (AIV) in wild bird populations are crucial to understanding the ecology of this pathogen. The standard method has been AIV isolation in chorioallantoic sac (CAS) of specific-pathogen-free embryonating chicken eggs (ECE), but in one study, combined use of yolk-sac (YS) and chorioallantoic membrane inoculation routes increased the number of virus isolations. In addition, cell culture for AIV isolation has been used. Most recently, real-time reverse transcriptase (RRT)-PCR has been used to detect AIV genome in surveillance samples. The purpose of this study was to develop a diagnostic decision tree that would increase AIV isolations from wild bird surveillance samples when using combinations of detection and isolation methods under our laboratory conditions. Attempts to identify AIV for 50 wild bird surveillance samples were accomplished via isolation in ECE using CAS and YS routes of inoculation, and in Madin-Darby canine kidney (MDCK) cells, and by AIV matrix gene detection using RRT-PCR. AIV was isolated from 36% of samples by CAS inoculation and 46% samples by YS inoculation using ECE, isolated from 20% of samples in MDCK cells, and detected in 54% of the samples by RRT-PCR. The AIV was isolated in ECE in 13 samples by both inoculation routes, five additional samples by allantoic, and 10 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 56%. Allantoic inoculation and RRT-PCR detected AIV in 14 samples, with four additional samples by allantoic route alone and 13 additional samples by RRT-PCR. Our data indicate that addition of YS inoculation of ECE will increase isolation of AIV from wild bird surveillance samples. If we exclude the confirmation RT-PCR test, cost analysis for our laboratory indicates that RRT-PCR is an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without prescreening by RRT-PCR was most efficient and cost-effective if the samples had an expected high frequency of AIV.