We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations.